Method for performing Rubella assay

ABSTRACT

The present invention includes novel rubella assays employing a Rubella virus capture reagent and a solid phase material containing a reaction site comprising a polymeric cation substance. A test sample suspected of containing Rubella antibody may be contacted with the capture reagent to form a capture reagent/analyte complex. The complex is then contacted to the positively charged solid phase to attract, attach, and immobilize the capture reagent/analyte complex.

This is a continuation-in-part of U.S. application Ser. No. 375,029,filed Jul. 7, 1989, now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 150,278, filed Jan. 29, 1988, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to the field of binding assay devicesand methods. In particular, the present invention relates to novelmethods and products useful in the performance of a rubella immunoassay.

2. Description of Related Art

Various analytical procedures and devices are commonly employed inassays to determine the presence and/or concentration of substances ofinterest or clinical significance which may be present in biologicalliquids or other materials. Such substances are commonly termed"analytes" and can include antibodies, antigens, drugs, hormones, etc.

Immunoassay techniques take advantage of the mechanisms of the immunesystems of higher organisms, wherein antibodies are produced in responseto the presence of antigens which are pathogenic or foreign to theorganisms. These antibodies and antigens, i.e., immunoreactants, arecapable of binding with one another, thereby creating a highly specificreaction mechanism which can be used in vitro to determine the presenceor concentration of that particular antigen in a biological sample.

There are several known immunoassay methods using immunoreactants,wherein at least one of the immunoreactants is labeled with a detectablecomponent so as to be analytically identifiable. For example, the"sandwich" or "two-site" technique may involve the formation of aternary complex between an antigen and two antibodies. A convenientmethod of detecting complex formation in such a technique is to provideone labeled antibody and an unlabeled antibody bound to a solid phasesupport such that the complex can readily be isolated. In this example,the amount of labeled antibody associated with the solid phase isdirectly proportional to the amount of analyte in the test sample.

An alternative technique is the "competitive" assay. In one example of acompetitive assay, the capture mechanism again may use an antibodyattached to an insoluble solid phase, but a labeled analyte (rather thana labeled antibody) competes with the analyte present in the test samplefor binding to the immobilized antibody. Similarly, an immobilizedanalyte can compete with the analyte of interest for a labeled antibody.In these competitive assays, the quantity of captured labeled reagent isinversely proportional to the amount of analyte present in the sample.

Despite their great utility, there are disadvantages with such assaymethods. First, the heterogenous reaction mixture of liquid test sampleand soluble and insoluble assay reagents, can retard the kinetics of thereaction. In comparison to a liquid phase reaction wherein all reagentsare soluble, i.e. a homogeneous reaction mixture, the heterogenousreaction mixture can require longer incubation periods for equilibriumto be reached in the reaction mixture between the insoluble solid phasesystem, the free analyte in the test sample, the soluble labeledreagent, and the newly formed insoluble complex. Second, conventionalmethods of attaching binding members to the solid phase, such asadsorption of antibody to the solid phase, can produce a solid phasewhich will readily bind substances other than the analyte. This isreferred to as nonspecific binding and can interfere with the detectionof a positive result. Third, with conventional immobilization methods,separate batches of manufactured solid phase reagents can containvariable amounts of immobilized binding member.

With regard to the manufacture of solid phase devices for use in bindingassays, there are a number of assay devices and procedures wherein thepresence of an analyte is indicated by the analyte's binding to alabeled reagent and/or a complementary binding member that isimmobilized on a solid phase such as a dipstick, teststrip, flow-throughpad, paper, fiber matrix or other solid phase material. Such a specificbinding reaction results in a distribution of the labeled reagentbetween that which is immobilized upon the solid phase and that whichremains free. Typically, the presence or amount of analyte in a testsample is indicated by the extent to which the labeled reagent becomesimmobilized upon the solid phase.

The use of porous teststrips in the performance of specific bindingassays is also well-known. In a sandwich assay procedure, a test sampleis applied to one portion of the teststrip and is allowed to migratethrough the strip material by means of capillary action. The analyte tobe detected or measured passes through the teststrip material, either asa component of the fluid test sample or with the aid of an eluting orchromatographic solvent which can be separately added to the strip. Theanalyte is thereby transported into a detection zone on the teststripwherein an analyte-specific binding member is immobilized. The extent towhich the analyte becomes bound in the detection zone can be determinedwith the aid of a labeled analyte-specific binding member which may beincorporated in the teststrip or which may be applied separately to thestrip.

Examples of devices based upon these principles include those describedthe following patents and patent applications. Deutsch et al. describe aquantitative chromatographic teststrip device in U.S. Pat. Nos.4,094,647, 4,235,601 and 4,361,537. The device comprises a materialcapable of transporting a solution by capillary action. Different areasor zones in the strip contain the reagents needed to perform a bindingassay and to produce a detectable signal as the analyte is transportedto or through such zones. The device is suited for chemical assays aswell as binding assays which are typified by the binding reactionbetween an antigen and a complementary antibody.

Many variations on the device of Deutsch et al. have been subsequentlydisclosed. For example, Tom et al. (U.S. Pat. No. 4,366,241) disclose abibulous support with an immunosorbing zone, containing an immobilizedspecific binding member. The test sample is applied to the immunosorbingzone, and the assay result is read at the immunosorbing zone.

Weng et al. (U.S. Pat. Nos. 4,740,468 and 4,879,215) also describe ateststrip device and methods for performing a binding assay. The deviceis used with a test solution containing the test sample, suspected ofcontaining the analyte of interest, and a labeled specific bindingmember which binds to the analyte. The assays involve both animmobilized second binding member, which binds to the labeled bindingmember, and an immobilized analog of the analyte, which removes unboundlabeled binding member from the assay system. Greenquist et al. (U.S.Pat. Nos. 4,806,311 and 4,806,312) describe a layered assay device forperforming binding assays similar to those of Weng et al., wherein afirst immobilized reagent such as an analyte-analog is used to removeunbound materials from the reaction mixture prior to the passage of thereaction mixture passage to a subsequent detection layer. Rosenstein(European Patent Office Publication No. 0 284 232) and Campbell et al.(U.S. Pat. Nos. 4,703,017) describe assay methods and devices forperforming specific binding assays, wherein the preferred detectablelabel is a colored particle consisting of a liposome containing a dye.Bahar, et al. (U.S. Pat. No. 4,868,108) describe an assay method anddevice for performing a specific binding assay, wherein the deviceinvolves a multizoned support through which test sample is transportedand an enzyme/substrate detection means. Eisinger et al. (U.S. Pat. No.4,943,522) describe an assay method and a device for performing specificbinding assays, using a multizoned large-pored lateral flow membranethrough which test sample is transported by capillary action.

Ullman et al. (European Patent Application No. 87309724.0; PublicationNo. 0 271 204) is related to the previously described Weng et al.patents (U.S. Pat. Nos. 4,740,468 and 4,879,215). Ullman et al. describethe preparation of a test solution containing an analyte-analog and atest sample suspected of containing the analyte. The test solution iscontacted to a bibulous material having two sequential binding sites:the first binding site containing a specific binding pair member capableof binding the analyte and the analyte-analog, the second binding sitecapable of binding that analyte-analog which is not bound at the firstbinding site.

Cerny E. (International Application No. PCT/US85/02534; Publication No.WO 86/03839) describes a binding assay wherein a test solution,containing the test sample and a labeled test substance, is allowed todiffuse through a solid phase to provide a measurable diffusion pattern.The resultant diffusion pattern has a diameter which is greater than thediameter of the diffusion pattern of the labeled test substance alone.

Zuk et al. (U.S. Pat. No. 4,956,275) describe a method and device fordetecting an analyte by means of a sensor apparatus. An analyte-relatedsignal is measured at two or more sites on the assay device by means ofthe sensor apparatus, and the mathematical relationship between themeasurements provides a value (e.g., difference, ratio, slope, etc.)which is compared against a standard containing a known amount ofanalyte.

Hochstrasser (U.S. Pat. No. 4,059,407) discloses a dipstick device whichcan be immersed in a biological fluid for a semi-quantitativemeasurement of the analyte in the fluid. The semi-quantitativemeasurement of the analyte is accomplished by using a series ofreagent-containing pads, wherein each pad in the series will produce adetectable color (i.e., a positive result) in the presence of anincreasing amount of analyte. Also of interest in the area of dipstickdevices are U.S. Pat. Nos. 3,802,842, 3,915,639 and 4,689,309.

Grubb et al. (U.S. Pat. No. 4,168,146) describe the use of a porousteststrip material to which an antigen-specific antibody is immobilizedby covalent binding to the strip. The teststrip is immersed in asolution suspected of containing an antigen, and capillary migration ofthe solution up the teststrip is allowed to occur. As the antigen movesup the teststrip it binds to the immobilized antigen-specific antibody.The presence of antigen is then determined by wetting the strip with asecond antigen-specific antibody to which a fluorescent or enzyme labelis covalently bound. Quantitative testing can be achieved by measuringthe length of the strip that contains bound antigen. Variations on sucha teststrip are disclosed in U.S. Pat. No. 4,435,504 which employs a twoenzyme indicator system; U.S. Pat. No. 4,594,327 which discloses theaddition of a binding agent to whole blood samples which causes the redblood cells to aggregate at the area of the strip adjacent to theair/liquid interface; and U.S. Pat. No. 4,757,004 which discloses ameans for controlling the shape of the fluid front migrating along theteststrip. The assay principle is further described in Zuk et al.,Enzyme Immunochromatography--A Quantitative Immunoassay Requiring NoInstrumentation, Clinical Chemistry, 31(7): 1144-1150, 1985.

Further examples of strip-type diagnostic devices include the following.Swanson et al. (EP 088 636) describe an apparatus for the quantitativedetermination of an analyte involving a fluid-permeable solid mediumcontaining a predetermined number of successive spaced reaction zones.The reaction zones include a reactant capable of reacting with theanalyte to produce a detectable signal; the greater the number of zonesproducing a detectable signal, the greater the amount of analyte in thetest sample. Freisen et al. (U.S. Pat. No. 4,861,711) describe asheet-like diagnostic device containing several functional sectorsthrough which the sample must pass. At least one of the sectors includesan immobilized reagent having a biological affinity for the analyte oran analyte complex.

Gordon et al. (U.S. Pat. No. 4,956,302) describe a teststrip devicecharacterized by having the analyte, test sample and/or eluting solventmigrate through the device in a single direction, thereby sequentiallycontacting reagent-containing zones or detection zones. Gordon et al.(U.S. Pat. No. 4,960,691) describe a device that includes one or morebounded pathways to direct the migration of the analyte, test sampleand/or eluting solvent through the reagent-containing zones anddetection zones in a predetermined order.

A variety of binding methods have been used to remove an analyte from atest solution. Bolz et al. (U.S. Pat. No. 4,020,151) describe asolid-phase assay for the quantitation of antigens or antibodies in atest sample. The sample antigen or antibody is adsorbed directly onto asolid support surface, such as anion exchange resin, and the support isthen exposed to a labeled specific binding member that isimmunologically reactive with the sample antigen or antibody.

Schick et al. (U.S. Pat. No. 4,145,406) describe the use of an ionexchange adsorbent to non-specifically bind protein. Marshall et al.(U.S. Pat. No. 4,211,763) describe a method for determining thyroidfunction involving an anion exchange resin to bind protein and form anagglomerate. Tabb et al. (U.S. Pat. No. 4,362,697) describe a testdevice involving the use of a copolymer of vinyl pyrrolidone as anenhancer substance. Giegel et al. (U.S. Pat. No. 4,517,288) describe amethod for conducting a ligand assay requiring the adsorption orimmunological binding of an analyte-specific binding member to a porousmedium, followed by the application of the analyte to the porous medium.

Other assay methods involve the use of auxiliary specific bindingmembers. Tanswell et al. (U.S. Pat. No. 4,624,930) describe a processfor determining the presence of a polyvalent antigen by incubating theantigen with three receptors; a first and a third receptor which bind tothe antigen and a second receptor, bound to a solid support, whichspecifically binds to the first receptor. Valkirs et al. (U.S. Pat. No.4,727,019) describe a method and device for ligand-receptor assays, asin Tanswell et al., wherein an anti-receptor (e.g., avidin) isimmobilized on a porous member and binds to a receptor (e.g., ananalyte-specific antibody bound to biotin) which is bound to the targetligand. Wolters et al. (U.S. Pat. No. 4,343,896) describe the use ofancillary specific binding members to prepare or complete detectablecomplexes, i.e., the use of a third antibody in a binding assay tocomplete a detectable analyte-binding member complex. W. Georghegan(U.S. Pat. No. 4,880,751) describes a method for preparing animmunoabsorption matrix by adsorbing the F(c) portion of a selected IgGmolecule onto a charged surface. Parikh et al. (U.S. Pat. No. 4,298,685)describe the use of a conjugate of biotin and an anti-analyte antibodytogether with an inert support bearing immobilized avidin. The specificbinding of the avidin and biotin components enables the immobilizationof the antibody on the inert support.

Alternative separation methods include the use of a magnetic solidphase, polymerization techniques and the formation of analyte complexeshaving characteristics different than the non-complexed analyte. Ullmanet al. (U.S. Pat. No. 4,935,147) describe a method for separatingcharged suspended non-magnetic particles from a liquid medium bycontacting the particles with charged magnetic particles and a chemicalreagent. The chemical reagent forms non-specific bonds between themagnetic and non-magnetic particles to produce a magnetic coaggregate. Amagnetic field gradient is applied to the reaction container toconcentrate the coaggregate to one part of the container, and the liquidmedium is then decanted.

Longoria et al. (U.S. Pat. No. 4,948,726) describe an assay methodinvolving the reaction of antigen and antibody molecules to form anantigen/antibody complex that uniquely exhibits an ionic charge that isdifferent from the ionic charges of the individual molecules. A filterpaper matrix is then chosen for its unique affinity for theantigen/antibody complex. Milburn et al. (U.S. Pat. No. 4,959,303)describe an assay wherein antigen from a test sample and an antibodyspecific for the antigen are incubated under conditions sufficient forthe antibody to bind to the support when the antigen is bound to theantibody. Bolz et al. (U.S. Pat. No. 4,020,151) describe a method ofimmobilizing the analyte molecule itself directly upon a solid support.Del Campo (U.S. Pat. No. 4,990,442) describes an assay involving thebinding of the analyte itself directly to an amphillic support byhydrogen bonding. Lyle et al. (European Application, Publication No.281,390) describe the preferential immobilization of polynucleotidesover olignucleotides to a polycationic support. Pronovost et al.(European Application, Publication No. 363,109) describe the separationof chlamydial or gonococcal antigen from a specimen using a positivelycharged solid support.

Vandekerckhove (U.S. Pat. No. 4,839,231) describes a two-stage, proteinimmobilization process involving an initial separation or isolation oftarget proteins in a gel, such as a polyacrylamide electrophoresis gel,followed by the transfer of those isolated proteins to the surface of acoated support for immobilization. The coated support is prepared bycontacting a chemically inert support material (which material bearsnegatively charged groups) with a solution of either polyvinylpyridineor polybrene (which polymer bears positively charged groups). Thecapacity of the positively charged polymer to form ionic linkages withthe negatively charged groups of the support material results in theformation of an insoluble polymeric film on the support.

Monji et al. (U.S. Pat. No. 4,780,409) describe a reactant conjugated toa temperature-sensitive or salt-sensitive polymer which will precipitatefrom a test solution when the temperature or salt concentration of thatsolution is adjusted to an appropriate level. Marshall (U.S. Pat. No.4,530,900) describes a reactant conjugated to a soluble polymer, whereinthe polymer is rendered insoluble for removal from solution and isphysically removed from the test solution by filtration orcentrifugation. Marshall discloses two means by which thisreactant-polymer conjugate is rendered insoluble: the lowering of the pHof the solution or the addition of a salt as in Monji et al. Marshallgoes on to describe that the insolubilized conjugate is thenprecipitated, removed from the test solution and finally resolubilizedto form a second solution prior to the detection of analyte.

As will be appreciated from the review of the background art, there issignificant activity in the teststrip field. There is a growing demandfor devices that require few or no manipulative steps to perform thedesired assay, for devices that can be used by relatively untrainedpersonnel, and for devices that provide results which are minimallyaffected by variations in the manner in which the assay is performed.Further considerations are the ease with which the resultant detectionsignal may be observed as well as the ease with which any signalsubstance immobilized at the detection site can be distinguished fromthe signal substance which passed through the detection site. Inaddition, a device manufacturing format has long been sought which willenable the production of a "generic" device, i.e., an assay device forwhich the capacity of use is defined by the reagents used in theperformance of the assay rather than the reagents used in themanufacture of the device.

SUMMARY OF THE INVENTION

The present invention provides novel a assay method for determining thepresence or amount of rubella antibody in a test sample. In oneembodiment, the assay involves a capture reagent, a capture reagent,comprising a Rubella virus; an indicator reagent, comprising a specificbinding member for Rubella antibody and a detectable label; and a solidphase material containing a reaction site comprising a polymeric cationsubstance thereby imparting a net positive charge to the solid phase.The solid phase is contacted with the capture reagent and the testsample, whereby the polymeric cation of the solid phase attracts andattaches to the Rubella virus capture reagent, thereby immobilizing thecapture reagent and complexes thereof. The solid phase is then contactedwith the indicator reagent, thereby immobilizing the indicator reagenton the solid phase in proportion to the amount of Rubella antibodypresent in the test sample. The indicator reagent associated with thesolid phase is then detected to determine the presence or amount of theanalyte in the test sample.

The present invention also enables the production of a generic solidphase device for use in specific binding assays. Assay procedures formany different analytes can use the same solid phase material whichcontains a predetermined zone of cationic capture polymer rather than animmobilized binding member capable of binding only a specific analyte asfound in conventional flow-through and teststrip devices.

The present invention provides two major advancements to the field ofspecific binding assays: a) the use of liquid phase kinetics facilitatesthe formation of a complex from the homogeneous mixture of analyte andassay reagent specific binding members, and b) the ion-capture techniqueincreases the potential number of complexes that can be immobilized on asolid support. If the advantages of liquid phase kinetics are notsought, the present invention also provides an efficient method ofimmobilizing binding members on a solid phase through a method otherthan absorption, adsorption or covalent binding.

DETAILED DESCRIPTION OF THE INVENTION

The assay methods and reagents of the present invention can be used in avariety of immunoassay formats. The present invention, however, is notlimited to immunoreactive assays. Any assays using specific bindingreactions between the analyte and assay reagents can be performed.

The present invention is particularly directed to the performance of abinding assay for the detection of rubella antibody present in a testsample. The assay is based upon the unexpected discovery that theRubella virus can be preferentially captured on a polycation-treatedsolid phase material and separated from free binding members present inthe test solution, thereby allowing the use of the Rubella virus as anion-capture reagent in binding assays for anti-Rubella antibody.

The assay reagents can include any suitable solid phase materialcontaining a cationic capturing zone, Rubella viruses and an indicatoragent, such as labeled anti-human IgG or IgM antibodies. Following theincubation of the test sample and the Rubella viruses, the test solutionis contacted to the cationic capturing zone wherein the reaction betweenthe polycationic substance and the Rubella viruses results in thecapture of the Rubella viruses upon the solid phase material. Thus, thebinding of the Rubella antibodies in the test sample to the Rubellaviruses results in the indirect immobilization of the Rubella antibodiesupon the solid phase. The immobilized complex can then be contacted toan indicator reagent which directly or indirectly binds to the Rubellaantibodies to form a sandwich complex. The means for the detection orquantitation of the analyte is dependent upon the nature of the label inthe indicator reagent.

Definitions

The following definitions are applicable to the present invention.

The term "specific binding member", as used herein, refers to a memberof a specific binding pair, i.e., two different molecules where one ofthe molecules through chemical or physical means specifically binds tothe second molecule. The complementary members of a specific bindingpair may also be referred to as a ligand and a receptor. In addition tothe well-known example of the antigen and antibody specific bindingpair, alternative specific binding pairs are exemplified by thefollowing: biotin and avidin, carbohydrates and lectins, complementarynucleotide sequences (including probe and capture nucleic acid sequencesused in DNA hybridization assays to detect a target nucleic acidsequence), complementary peptide sequences (including those formed byrecombinant methods), effector and receptor molecules, hormone andhormone binding protein, enzyme cofactors and enzymes, enzyme inhibitorsand enzymes, and the like. Furthermore, specific binding pairs caninclude members that are analogs of the original specific bindingmember. For example, a derivative or fragment of the analyte (ananalyte-analog) can be used so long as it has at least one epitope incommon with the analyte. Immunoreactive specific binding members includeantigens, haptens, antibodies, and complexes thereof including thoseformed by recombinant DNA methods or peptide synthesis. An antibody canbe a monoclonal or polyclonal antibody, a chimeric antibody, arecombinant protein or a mixture(s) or fragment(s) thereof, as well as amixture of an antibody and other specific binding members. The detailsof the preparation of such antibodies and their suitability for use asspecific binding members are well-known to those skilled-in-the-art.

The term "hapten", as used herein, refers to a partial antigen ornon-protein binding member which is capable of binding to an antibody,but which is not capable of eliciting antibody formation unless coupledto a carrier protein.

The term "test sample", as used herein, refers to virtually any liquidsample. The test sample can be derived from any desired source, such asa physiological fluid, for example, blood, saliva, ocular lens fluid,cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous,synovial fluid, peritoneal fluid, amniotic fluid or the like. The liquidtest sample can be pretreated prior to use, such as preparing plasmafrom blood, diluting viscous liquids, etc. Methods of pretreatment canalso involve separation, filtration, distillation, concentration,inactivation of interfering components, and the addition of reagents.Besides physiological fluids, other liquid samples such as water, foodproducts and the like can be used. In addition, a solid test sample canbe used once it is modified to form a liquid medium.

The term "analyte", as used herein, refers to the substance to bedetected in or separated from the test sample by means of the presentinvention. The analyte can be any substance for which there exists anaturally occurring specific binding member or for which a specificbinding member can be prepared. In addition, the analyte may bind tomore than one specific binding member. "Analyte" also includes anyantigenic substances, haptens, antibodies, and combinations thereof. Theanalyte can include, but is not limited to, a protein, a peptide, anamino acid, a hormone, a steroid, a vitamin, a drug including thoseadministered for therapeutic purposes as well as those administered forillicit purposes, a bacterium, a virus, and metabolites of or antibodiesto any of the above substances.

The term "analyte-analog", as used herein, refers to a substance whichcross-reacts with a binding member specific for the analyte, althoughthe analyte-analog may react with the binding member to a greater or alesser extent than does the analyte itself. The analyte-analog caninclude a modified analyte as well as a fragmented or synthetic portionof the analyte molecule so long as the analyte-analog has at least oneepitopic site in common with the analyte of interest.

The term "label", as used herein, refers to any substance which directlyor indirectly attaches to a specific binding member and which is capableof producing a signal that is detectable by visual or instrumentalmeans. Various suitable labels for use in the present invention caninclude chromogens; catalysts; fluorescent compounds; chemiluminescentcompounds; radioactive labels; direct visual labels including colloidalmetallic and non-metallic particles, dye particles, enzymes orsubstrates, or organic polymer latex particles; liposomes or othervesicles containing signal producing substances; and the like.

A large number of enzymes suitable for use as labels are disclosed inU.S. Pat. No. 4,275,149, columns 19-23, the disclosure of which ishereby incorporated by reference. For example, an enzyme/substratesignal producing system useful in the present invention involves theenzyme alkaline phosphatase and the substrate nitro bluetetrazolium-5-bromo-4-chloro-3-indolyl phosphate or a derivative oranalog thereof.

In an alternative signal producing system, the label can be afluorescent compound where no enzymatic manipulation of the label isrequired to produce a detectable signal. Fluorescent molecules such asfluorescein, phycobiliprotein, rhodamine and their derivatives andanalogs are suitable for use as labels in this system.

In an especially preferred embodiment of the present invention, avisually detectable, colored particle can be used as the label componentof the indicator reagent, thereby providing for a direct colored readoutof the presence or concentration of the analyte in the sample withoutthe need for the addition of other signal producing reagents. Materialsfor use as the colored particles are colloidal metals, such as gold, anddye particles as disclosed in U.S. Pat. Nos. 4,313,734 and 4,373,932 thedisclosures of which are hereby incorporated by reference. Thepreparation and use of non-metallic colloids, such as colloidal seleniumparticles, are disclosed in U.S. Pat. No. 4,954,452 the disclosure ofwhich is hereby incorporated by reference. The use of colloidal particlelabels in immunochromatography is disclosed in co-owned U.S. Pat. No.5,120,643 which is hereby incorporated by reference. Organic polymerlatex particles for use as labels are disclosed in co-owned andcopending U.S. patent application Ser. No. 248,858, filed Sep. 23, 1988now U.S. Pat. No. 5,252,459 the disclosure of which is herebyincorporated by reference.

The term "signal producing component", as used herein, refers to anysubstance capable of reacting with the analyte or another assay reagentto produce a reaction product or signal that indicates the presence oramount of the analyte and that is detectable by visual or instrumentalmeans. "Signal production system", as used herein, refers to the groupof assay reagents that are used to produce the desired reaction productor signal. For example, one or more signal producing components can beused to react with a label and generate the detectable signal. Forexample, when the label is an enzyme, amplification of the detectablesignal is obtained by reacting the enzyme with one or more substrates oradditional enzymes to produce a detectable reaction product.

The term "indicator reagent", as used herein, refers to a specificbinding member which is attached or which becomes attached to a label.The indicator reagent produces a detectable signal at a level relativeto the amount of an analyte in the test sample. Generally, the indicatorreagent is detected or measured after it is captured on the solid phasematerial, but the unbound indicator reagent can also be measured todetermine the result of an assay.

The specific binding member of the indicator reagent is capable ofbinding either to the analyte as in a sandwich assay, to the capturereagent as in a competitive assay, or to an ancillary specific bindingmember to complete a detectable complex. The label, as described above,enables the indicator reagent to produce a detectable signal that isrelated to the presence or amount of analyte in the test sample. Thespecific binding member component of the indicator reagent enables theindirect binding of the label to the analyte, to an ancillary specificbinding member or to the capture reagent. The selection of a particularlabel is not critical, but the label will be capable of generating adetectable signal either by itself, such as a visually detectable signalgenerated by colored organic polymer latex particles, or in conjunctionwith one or more additional signal producing components, such as anenzyme/substrate signal producing system. A variety of differentindicator reagents can be formed by varying either the label or thespecific binding member; it will be appreciated by oneskilled-in-the-art that the choice involves consideration of the analyteto be detected and the desired means of detection.

As mentioned above, the label can become attached to the specificbinding member during the course of the assay. For example, abiotinylated anti-analyte antibody may be reacted with a labeledstreptavidin molecule. Any suitable combination of binding members andlabels can be used.

The term "capture reagent", as used herein, refers to an unlabeledspecific binding member which is attached to a charged substance. Theattachment of the components is essentially irreversible and can includecovalent mechanisms. The specific binding member can be a smallmolecule, such as a hapten or small peptide, so long as the attachmentto the charged substance does not interfere with the binding member'sbinding site. The binding member component of the capture reagent isspecific either for the analyte as in a sandwich assay, for theindicator reagent or analyte as in a competitive assay, or for anancillary specific binding member, which itself is specific for theanalyte.

The charged substance component of the capture reagent can includeanionic and cationic monomers or polymers. For example, anionic polymersinclude polyglutamic acid (PGA), anionic protein or derivatized proteinsuch as albumin, anionic polysaccharides such as heparin or alginicacid, polyaspartic acid, polyacrylic acid, and polyamino acids having anet negative charge at a pH appropriate for the specific bindingreaction (such as a pH in the range of 4 to 10.) Furthermore, thespecific binding member can be joined to more than one charged monomeror polymer to increase the net charge associated with the capturereagent.

The novel capture reagents of the present invention are used tofacilitate the observation of the detectable signal by substantiallyseparating the analyte and/or the indicator reagent from other assayreagents and the remaining test sample components. In its mostadvantageous use, the capture reagent is reacted with the test sampleand assay reagents in a homogeneous reaction mixture. Following theformation of the desired specific binding member complexes, thecomplexes involving a capture reagent are removed from the homogeneousreaction mixture by contacting the homogeneous reaction mixture to asolid phase that is oppositely charged with respect to the charge of thecapture reagent.

In one embodiment of the present invention, a negatively charged capturereagent can be prepared by conjugating the selected specific bindingmember to one or more activated polymeric anionic molecules andconjugate bases thereof represented by the general formula: ##STR1##wherein n is about 10 to about 500; z is about 1 to about 6; W is chosenfrom H⁺, Na⁺, K⁺, Li⁺, amine salts such as H⁺ NR₃, and derivativesthereof; and X is virtually any reactive group or moiety having areactive group that enables the chemical binding of the specific bindingmember and the polymer. "X" can be an amine-reactive group or moiety, athiol-reactive group or moiety, or a thiol group or moiety representedby --A--SH wherein A is a spacer arm. For example, a specific bindingmember having an amino group can be conjugated to an activated PGAanionic molecule having an amine-reactive moiety. The amine-reactivemoieties enable the binding of the activated polymer to an amino groupon a specific binding member and include, but are not limited to, thoserepresented by the following formulas: ##STR2## wherein m is two orthree, R' is a sulfur stabilizer and R" is an aliphatic or aryl group.

Sulfur stabilizers include, but are not limited to, 2-pyridyl, 4-pyridyland 5-nitro-2-pyridyl groups. "A" represents a spacer of about one toabout thirty atoms including, but not limited to, carbon, nitrogen,sulfur and oxygen atom chains and combinations thereof such aspolyether, polymethylene and polyamide, as well as aromatic spacers suchas phenylthiocarbamyl.

Alternatively, a specific binding member having a thiol group can beconjugated to an activated polymer having a thiol-reactive moiety. Thethiol-reactive moieties include, but are not limited to, thoserepresented by the following formulas: ##STR3## wherein A is a spacer ofabout one to about thirty atoms as described above. In yet anotheralternative, a specific binding member having a thiol-reactive group canbe linked to an activated polymer having a thiol moiety such as --A--SH.

Typically, the negatively charged capture reagents of the followingExamples were formed by reacting the desired specific binding memberwith an activated PGA molecule having modified terminal amino groups.Briefly, the modification method involved: 1) dissolving the PGA in asolvent (e.g., a water miscible aprotic solvent such as dioxane,dimethylformamide, 1-methyl-2-pyrrolidinone and dimethyl sulfoxide); 2)adding a proton absorbing reagent (e.g., 4-methyl morpholine) in theamount of about one equivalent per titratable carboxylic acid; 3) addingabout a 2 to about a 100 molar excess of an amine-reactive modificationreagent (e.g., 1,4-phenylene diisothiocyanate dissolved indimethylformamide); 4) reacting the mixture; and 5) removing theunreacted amine-reactive modification reagent. Suitable proton absorbingreagents include alkali metal hydroxides such as sodium hydroxide,potassium hydroxide or lithium hydroxide, and tertiary amines such as4-methyl morpholine and triethylamine.

The polymeric anionic molecule or the specific binding member willinclude one or more amino, carboxyl or thiol groups, or can be activatedby the incorporation of an amino, carboxyl or thiol group, therebyenabling the chemical cross-linking of the specific binding member withthe polymeric anionic molecule. "Activated species" refer to specificbinding members and polymeric anionic molecules which contain a reactivegroup through the incorporation of a cross-linking or other activatingagent. The amine-reactive modification reagents are a subclass of thosereagents used to "activate" a specific binding member or polymericanionic molecule, i.e., to prepare the specific binding member or thepolymeric anionic molecule for chemical cross-linking. Activating agentsalso include thiol introducing agents such as the thiolanes (such as2-iminothiolane), succinimidyl mercaptoacetates (such asN-succinimidyl-S-acetylmercaptoacetate), and disulfide compounds whichare subsequently reduced to a thiol. The thiol introducing agents can beused to activate specific binding members and solid phase materials fortheir subsequent reaction with a thiol-reactive group.

Amine-reactive modification reagents include, but are not limited to,bifunctional crosslinking or coupling agents, such as succinic anhydrideanalogs, iminothiolane analogs, homobifunctional reagents andheterobifunctional reagents, which enable the chemical cross-linking ofthe specific binding member and the polymeric anionic molecule. Examplesof homobifunctional reagents can be represented by the formula X--A--Xwherein X is an amine-reactive group and A is a spacer of about one toabout thirty atoms. Examples of heterobifunctional reagents can berepresented by the formula X--A--Y, wherein X is an amine-reactivegroup, Y is a thiol-reactive moiety, a thiol moiety or a thiol precursorand A is a spacer of about one to about thirty atoms as described above.Proteinaceous specific binding members with cysteine residues at theprotein's active site can have their activity decreased by the additionof a coupling agent, therefore the cysteine residues in the active sitemust be protected, by means known in the art, prior to reacting theprotein with the coupling agent.

The term "coupling agent", as used herein, includes bifunctionalcrosslinking or coupling agents, i.e., molecules containing two reactivegroups or "ends", which may be tethered by a spacer. The reactive endscan be any of a variety of functionalities including, but not limitedto: amino reacting ends such as N-hydroxysuccinimide (NHS) activeesters, imidoesters, aldehydes, epoxides, sulfonyl halides, isocyanate,isothiocyanate, and nitroaryl halides; and thiol reacting ends such aspyridyl disulfides, maleimides, thiophthalimides, and active halogens.The heterobifunctional crosslinking reagents have two different reactiveends, e.g., an amino-reactive end and a thiol-reactive end, whilehomobifunctional reagents have two similar reactive ends, e.g.,bismaleimidohexane (BMH) which permits the cross-linking ofsulfhydryl-containing compounds, and NHS homobifunctional crosslinkerssuch as disuccinimidyl suberate (DSS) as well as the water solubleanalogs, sulfo-NHS esters (Pierce 1989 Handbook and General Catalog;Pierce, Rockford, Ill.).

Other commercially available homobifunctional cross-linking reagentsinclude, but are not limited to, the imidoesters such as dimethyladipimidate dihydrochloride (DMA);

dimethyl pimelimidate dihydrochloride (DMP); and dimethyl suberimidatedihydrochloride (DMS). The iminothiolane analogs can be represented bythe general formula: ##STR4## wherein A is a spacer of about 1 to about5 atoms, e.g., 2-iminothiolane (Traut's Reagent.) Commercially availableheterobifunctional reagents suitable for use in the present inventioninclude, but are not limited to, maleimido-NHS active esters couplingagents such as m-maleimidobenzoyl-N-hydroxy-succinimide ester (MBS);succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC);succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB) and derivativesthereof, including sulfosuccinimidyl derivatives such assulfosuccinimidyl 4-(N-maleimido-methyl) cyclohexane-1-carboxylate(sulfo-SMCC); m-maleimidobenzoyl-sulfosuccinimide ester (sulfo-MBS) andsulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB) (Pierce).Other suitable heterobifunctional reagents include commerciallyavailable active halogen-NHS active esters coupling agents such asN-succinimidyl bromoacetate andN-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB) and thesulfosuccinimidyl derivatives such assulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) (Pierce).Another group of coupling agents is the heterobifunctional and thiolcleavable agents such as N-succinimidyl 3-(2-pyridyidithio)propionate(SPDP) (Pierce).

Yet another group of coupling agents includes the extended lengthheterobifunctional coupling agents described in co-owned and copendingU.S. patent applications Ser. Nos. 254,288 (filed Oct. 11, 1988) and114,930 (filed Oct. 30, 1987) the disclosures of which are herebyincorporated by reference. The extended length heterobifunctionalcoupling agents include maleimido-NHS active ester reagents wherein thespacer is represented by the formula: ##STR5## wherein the amino acid isa substituted or unsubstituted amino acid, having from three to tencarbon atoms in a straight chain; n is from one to ten; and R is analkyl, cycloalkyl, alkyl-cycloalkyl or an aromatic carboxylic ring. Theterm alkyl-cycloalkyl includes alkyl groups linked to cycloalkyl ringstructures where the alkyl group links the cycloalkyl to a maleimide orcarbonyl group. The term alkyl includes straight or branched alkylgroups, preferably lower alkyl groups having from one to six carbonatoms.

If a spacer is present, the spacer can be any molecular chain that isnon-reactive, stable and non-binding to the analyte or other specificbinding members with which it will be used. The length of the spacer canbe varied and can range from the size of a single atom, to the sizesdisclosed in U.S. Pat. No. 5,002,883 and U.S. patent application Ser.No. 114,930, now abandoned, or larger.

The choice of the amine-reactive modification reagent, thiol introducingagent or other activating agent is not critical, but oneskilled-in-the-art will know of suitable or preferred agents for usewith the particular polymeric anionic molecule and specific bindingmember to be used in the diagnostic assay. Therefore, it will beappreciated by those skilled-in-the-art that the coupling agent oractivating agent used in a given assay will generally be determinedempirically.

Suitable thiol-reactive moieties of the heterobifunctional reagentsinclude, but are not limited to, those represented by the followingformulas: ##STR6## Suitable thiol precursor moieties include, but arenot limited to, those represented by the following formulas: ##STR7##Suitable amine-reactive moieties include, but are not limited to, thoserepresented by the following formulas: ##STR8## wherein m is 2 or 3, R'is a sulfur stabilizer, as described above, and R" is an aliphatic oraryl group.

In yet another embodiment of the present invention, a specific bindingmember having an amine-reactive group (e.g., an activated specificbinding member) can be conjugated to a terminal amino group of thepolymeric anionic molecule. Briefly, an example of a conjugationprocedure involves: 1) dissolving PGA in a solvent (e.g., a watermiscible aprotic solvent such as dioxane, dimethylformamide,1-methyl-2-pyrrolidinone and dimethyl sulfoxide); 2) adding a protonabsorbing reagent (e.g., an alkali metal hydroxide such as sodiumhydroxide, potassium hydroxide, or lithium hydroxide, or a tertiaryamine such as 4-methyl morpholine or triethylamine) in the amount ofabout one equivalent per titratable carboxylic acid; 3) adding about a 2to about a 100 molar excess of amine-reactive specific binding member(e.g., phosgene-activated phenylcyclidine orphenylcyclidine-4-chloroformate); 4) reacting the mixture and 5)removing the unreacted amine-reactive specific binding member. Examplesof suitable amine-reactive groups on specific binding members include,but are not limited to, the following: ##STR9## wherein A is a spacer ofabout one to about thirty atoms as described above, m is two or three,R' is a sulfur stabilizer and R" is an aliphatic or aryl group.

An example of the preparation of a negatively charged capture reagentinvolves the reaction of a specific binding member (SBM) having an aminogroup and an activated PGA having an amine-reactive moiety. Theresulting reaction and reaction product can be illustrated as follows:##STR10##

In yet another embodiment of the present invention, a preferred anionicpolymer for use in the capture reagent is carboxymethylamylose (CMA) dueto its particular performance in various immunoassay configurations. Theimproved performance of capture reagents containing CMA can beattributed to the higher avidity of the CMA capture reagent for thecationic solid phase. This attribute is particularly advantageous in atwo step sandwich assay format wherein a polyanion is used to blocknonspecific binding of the indicator reagent to the cationi solid phase.

The term "ancillary specific binding member", as used herein, refers toany member of a specific binding pair which is used in the assay inaddition to the specific binding members of the capture reagent and theindicator reagent. For example, in an assay an ancillary specificbinding member may bind the analyte to a second specific binding memberto which the analyte itself could not attach, or as in an inhibitionassay the ancillary specific binding member may be a reference bindingmember. One or more ancillary specific binding members can be used in anassay.

The term "solid phase", as used herein, refers to any material which isinsoluble, or can be made insoluble by a subsequent reaction. The solidphase can be chosen for its intrinsic charge and ability to attract thecapture reagent, e.g., methylated wool, nylons, and special glasseshaving a positive charge. Alternatively, the solid phase can bepretreated with and retain a charged substance that is oppositelycharged with respect to the charged substance of the capture reagent.For example, an anionic substance can be bound to a specific bindingmember to form the capture reagent, and a cationic substance can beapplied to and retained by the solid phase, or vice versa.

Natural, synthetic, or naturally occurring materials that aresynthetically modified, can be used as the cationic substance. A widevariety of proprietary polycations are available including tertiary andquaternary ammonium compounds and derivatives of ammonium compounds.Such polycationic materials include, but are not limited to,hexadimethrine bromide (Polybrene®; Sigma Chemical Company, St. Louis,Mo.), GAFQuat™ quaternary ammonium compounds (GAF Corporation, Wayne,N.J., 07470), diethylaminoethyl-dextran (Sigma), Merquat-100® compound(a cationic homopolymer of dimethyidiallylammonium chloride; CalgonCorporation, Pittsburgh, Pa.) and water soluble cellulose derivativessuch as diallyldimethylammonium chloride-hydroxyethyl cellulose polymer(Celquat® L-200 polymeric quaternary ammonium compounds and Celquat®H-100 polymeric compounds, National Starch & Chemical Corporation,Bridgewater, N.J., 08807).

It was unexpectedly discovered that the cationic homopolymer ofdimethyldiallylammonium chloride and other polycationic substanceshaving a nitrogen content above about 2% (exclusive of counter ion) areparticularly advantageous in preparing a solid phase that will undergowashing during the assay process. The use of such a polycationicsubstance to prepare a suitably charged solid phase resulted in a solidphase that could be subjected to a greater degree of manipulationwithout losing the capability to attract and retain the oppositelycharged capture reagent. It was determined that polycationic substanceshaving a nitrogen content above about 5% (exclusive of counter ion) weremore preferred and that substances having a nitrogen content above about10% (exclusive of counter ion) were most preferred.

An assay device based on the ion-capture technique can have manyconfigurations, several of which are dependent upon the material chosenas the solid phase. In various device embodiments, the solid phase mayinvolve polymeric or glass beads, microparticles, magnetic particles,tubes, sheets, plates, slides, wells, tapes, test tubes, layered filmsor the like, or any other material which has an intrinsic charge orwhich can retain a charged substance.

The novel ion-capture devices of the present invention involve a solidphase made of any suitable porous material. By "porous" is meant thatthe material is one through which the test sample can easily pass bycapillary or wicking action and includes both bibulous and non-bibuloussolid phase materials. For example, the solid phase can include afiberglass, cellulose, or nylon pad for use in a flow-through assaydevice having one or more layers containing one or more of the assayreagents; a dipstick for a dip and read assay; a teststrip for wickingor capillary action (e.g., paper, nitrocellulose, polyethylene)techniques; or other porous or open pore materials well-known to thoseskilled-in-the-art (e.g., polyethylene sheet material as manufactured byPorex Technologies Corporation, Fairburn, Ga., USA).

Natural, synthetic, or naturally occurring materials that aresynthetically modified, can be used as a solid phase includingpolysaccharides, e.g., cellulose materials such as paper and cellulosederivatives such as cellulose acetate and nitrocellulose; silica;inorganic materials such as deactivated alumina, diatomaceous earth,MgSO₄, or other inorganic finely divided material uniformly dispersed ina porous polymer matrix, with polymers such as vinyl chloride, vinylchloride-propylene copolymer, and vinyl chloride-vinyl acetatecopolymer; cloth, both naturally occurring (e.g., cotton) and synthetic(e.g., nylon); porous gels such as silica gel, agarose, dextran, andgelatin; polymeric films such as polyacrylamide; and the like. The solidphase should have reasonable strength or strength can be provided bymeans of a support, and it should not interfere with the production of adetectable signal.

Preferred solid phase materials for flow-through assay devices includefilter paper such as a porous fiberglass material or other fiber matrixmaterials as well as isotropically porous materials such as apolyethylene pad. The thickness of the material used will be a matter ofchoice, largely based upon the properties of the sample or analyte beingassayed, e.g., the fluidity of the test sample.

To change or enhance the intrinsic charge of the solid phase, a chargedsubstance can be applied directly to the solid phase material or tomicroparticles which are then retained by a solid phase supportmaterial. Alternatively, coated microparticles alone can be used as thecharged solid phase by being retained in a column. By "retained" ismeant that the particles on or in the solid phase support material arenot capable of substantial movement to positions elsewhere within thesupport material. The particles can be selected by oneskilled-in-the-art from any suitable type of particulate material andinclude those composed of polystyrene, polymethylacrylate,polypropylene, latex, polytetrafluoroethylene, polyacrylonitrile,polycarbonate, glass or similar materials. The size of the particles isnot critical, although it is preferred that the average diameter of theparticles be smaller than the average pore size of the support materialbeing used.

Typically, the novel teststrip and flow-through devices employing theion-capture principles of the present invention are characterized byhaving the analyte, test sample and/or eluting solvent migrate throughthe device in a single direction, thereby sequentially contactingreagent-containing zones or detection zones. Alternatively, the noveldevices of the present invention may be configured such that the analytemigrates radially from the sample application site to thereagent-containing zones or detection zones. In yet another embodiment,the novel devices may include one or more bounded pathways to direct themigration of the analyte, test sample and/or eluting solvent through thereagent-containing zones and detection zones in a predetermined order.

Uses for Ion-Capture Reagents

In accordance with the disclosure of the present invention, a sandwichassay can be performed wherein the capture reagent involves ananalyte-specific binding member which has been bound to a chargedsubstance such as an anionic polymer. The capture reagent is contactedwith a test sample, suspected of containing the analyte, and anindicator reagent comprising a labeled analyte-specific binding member.The reagents can be mixed simultaneously or added sequentially, eithersingly or in combination to form a homogenous reaction mixture.

In the exemplary sandwich assay, a binding reaction results in theformation of a capture reagent/analyte/indicator reagent complex. Theresultant complex is then removed from the excess assay reagents andtest sample of the homogenous reaction mixture by means of a solid phasethat is either inherently oppositely charged with respect to the capturereagent or that retains an oppositely charged substance, for example acationic polymer. In the ion-capture assays, the oppositely chargedsolid phase attracts and attaches to the capturereagent/analyte/indicator reagent complex through the interaction of theanionic and cationic polymers. The complex retained on the solid phaseis then detected by examining the solid phase for the indicator reagent.If analyte is present in the sample, then label will be present on thesolid phase. The amount of label on the solid phase is proportional tothe amount of analyte in the sample. The only major limitation inherentin the sandwich assay is the requirement for the analyte to have asufficient size and appropriately orientated epitopes to permit thebinding of at least two specific binding members. Other sandwich assaysmay involve one or more ancillary specific binding members to bind theanalyte to the indicator reagent and/or capture reagent.

The present invention also can be used to conduct a competitive assay.In an exemplary competitive assay, the soluble capture reagent againincludes a specific binding member which has been attached to a chargedsubstance, such as an anionic polymer. The capture reagent is contacted,either sequentially or simultaneously, with the test sample and anindicator reagent that includes a second binding member which has beenlabeled with a signal generating compound. Either the capture reagentand analyte can compete in binding to the indicator reagent (e.g., thecapture reagent and analyte are antigens competing for a labeledantibody), or the indicator reagent and analyte can compete in bindingto the capture reagent (e.g., the indicator reagent is a labeled antigenwhich competes with the antigen analyte for binding to the antibodycomponent of the capture reagent). A competitive binding or displacementreaction occurs in the homogeneous mixture and results in the formationof capture reagent/analyte complexes and capture reagent/indicatorreagent complexes.

The resultant complexes are removed from the excess assay reagents andtest sample by contacting the reaction mixture with the oppositelycharged solid phase. The capture reagent complexes are retained on thesolid phase through the interaction of the oppositely charged polymers.The complexes retained on the solid phase can be detected via the labelof the indicator reagent. In the competitive assay, the amount of labelthat becomes associated with the solid phase is inversely proportionalto the amount of analyte in the sample. Thus, a positive test samplewill generate a negative signal. The competitive assay is advantageouslyused to determine the presence of small molecule analytes, such as smallpeptides or haptens, which have a single epitope with which to bind aspecific binding partner. Other competitive assays may involve one ormore ancillary specific binding members to bind the analyte to theindicator reagent and/or capture reagent.

For example, in an assay for theophylline, an anti-theophylline antibody(either monoclonal or polyclonal) can be conjugated with an anionicpolymer to form a soluble capture reagent, and a competition for bindingto that antibody can be established between labeled theophylline (i.e.,indicator reagent) and the unlabeled theophylline of the test sample.After incubation, the homogeneous mixture can be contacted to a solidphase which retains a cationic polymer coating. The attraction betweenthe oppositely charged ionic species of the capture reagent and thesolid phase serves to separate the immunocomplex from the reactionmixture. The signal from the indicator reagent can then be detected. Inthis example, increased theophylline levels in the test sample willresult in decreased signal generation associated with the solid phase.

In addition, the present invention can be used in an inhibition assay,such as the measurement of an antibody by inhibiting the detection of areference antigen. For example, the capture reagent can include anantibody/anionic polymer conjugate and the indicator reagent can be alabeled antibody. The test sample, suspected of containing an antibodyanalyte, is mixed with a reference antigen with which the capturereagent and indicator reagent can form a detectable sandwich complexthat can be immobilized upon the solid phase by the ion-capturereaction. The degree of inhibition of antigen uptake by the capturereagent is proportional to the amount of antibody analyte in the testsample, thus, as the concentration of the antibody analyte increases,the less reference antigen is available to complete the immobilizedsandwich complex.

In general, once complex formation occurs between the analyte and theassay reagents, the oppositely charged solid phase is used as aseparation mechanism: the homogeneous reaction mixture is contacted withthe solid phase, and the newly formed binding complexes are retained onthe solid phase through the interaction of the opposite charges of thesolid phase and the capture reagent. If the user is not concerned withliquid phase kinetics, the capture reagent can be pre-immobilized on thesolid phase to form a capture site.

The present invention can also be used for separating a substance from aliquid sample. For example, the capture reagent and solid phase can beused without an indicator reagent for the sole purpose of separating ananalyte from a test sample. Furthermore, the capture reagent can becontacted with a soluble second charged substance which is oppositelycharged with respect to the capture reagent. The second chargedsubstance is not retained on the solid phase prior to contacting thesample to the solid phase material, but it attracts and attaches to thecapture reagent such that the resultant assay complexes are retained onan oppositely charged solid phase.

When the complex of charged capture reagent and analyte (and/orindicator reagent) is contacted to the oppositely charged solid phase,the ionic attraction of the oppositely charged species governs theefficiency of the separation of the complex from the reaction mixture.The ionic attraction can be selected to provide a greater attractionthan the immunological attraction of antibody for antigen, particularlywhen multiple polycationic and polyanionic species are included in thecapture reagent and oppositely charged solid phase. A further advantageis that the "ion-capture" technique minimizes the nonspecific adsorptionof interfering substances onto the solid phase, thereby offeringimproved accuracy of analysis. The ion-capture technique thereby enablesthe performance of an assay having a highly specific separation method,minimal nonspecific binding, and high sensitivity.

In one embodiment of the present invention, it was discovered that theaddition of a nonspecific binding blocker reagent to the indicatorreagent resulted in an increase in the signal to noise ratio. It wasunexpectedly discovered that the nonspecific binding blocker could be afree polyanion even when the capture reagent used in the assay involveda polyanionic substance conjugated to a specific binding member. Itwould have been expected that the presence of a free or unboundpolyanion would prevent, or at least reduce, the immobilization of thecapture reagent on the solid phase. It was found, however, that thenonspecific blocker was more effective in inhibiting the direct,nonspecific binding of indicator reagent to the solid phase than it wasin reducing the attachment of the polyanionic capture reagent to thepolycationic solid phase. Suitable nonspecific binding blockers include,but are not limited to, dextran sulfate, heparin, carboxymethyl dextran,carboxymethyl cellulose, pentosan polysulfate, inositol hexasulfate andβ-cyclodextrin sulfate.

It was also discovered that the amount of polyanionic nonspecificbinding blocker added to the indicator reagent could be greater than theamount of polyanionic substance contained in the capture reagent. It wasfound that free polyanionic nonspecific binding blocker could be addedto the indicator reagent in amounts 40,000 times the amount ofpolyanionic substance used in the capture reagent. Generally, thepreferred amount of polyanionic blocker added to the indicator reagentis 50 to 14,000 times the amount of polyanionic substance used in thecapture reagent. For two step sandwich assays, the preferred amount ofpolyanionic blocker added to the indicator reagent is 1000 to 2000 timesthat contained in the capture reagent.

An appropriate range of use can be determined for each analyte ofinterest. For example, in an assay to detect thyroid stimulating hormone(TSH) wherein dextran sulfate was added to the indicator reagent as afree polyanionic nonspecific binding blocker, suitable amounts of freepolyanion ranged from 233 to 19,000 times that of the capture reagent,or about 0.1-8% dextran sulfate. As illustrated in the following Table,the preferred nonspecific binding blocker as well as the preferredamount of nonspecific binding blocker can be optimized for each analyteof interest.

    ______________________________________    Nonspecific Binding Blocker    in the Indicator Reagent    Analyte     Preferred     More Preferred    ______________________________________    % Dextran sulfate (MW 5,000)    (blocker/capture reagent, w/w)    TSH         0.1-8         0.5-2                (233-19,000)  (1,000-4,000)    T3          0.1-2         0.1-0.2                (2,000-40,000)                              (2,000-4,000)    % Carboxymethyl cellulose (MW 250,000)    (blocker/capture reagent, w/w)    hCG         0.01-0.25     0.025                (0.44-11)     (1.1)    HIV         0-0.2         0.05                (0-20,000)    (5,000)    ______________________________________

Moreover, it was discovered that the polyanionic nonspecific bindingblocker could be added to the assay as a separate reagent, or it couldbe included as free polyanion in the capture reagent, in an ancillarybinding member reagent, in a buffer reagent or in some other reagentused in the assay. For example, when free polyanion is included in thecapture reagent, it can enhance the signal to noise ratio byneutralizing interfering materials which are contained either in thetest sample itself or in the other assay reagents, or those which wereintroduced during the device manufacturing process. The following Tableillustrates some preferred amounts of nonspecific binding blocker fordifferent analytes of interest, wherein the free polyanion is containedin the capture reagent itself.

    ______________________________________    Nonspecific Binding Blocker    in the Capture Reagent    Analyte      Preferred    More Preferred    ______________________________________    % Dextran sulfate (MW 5,000)    (blocker/capture reagent, w/w)    Digoxin      0-0.004      0.004                 (0-222)      (222)    T3           0.004-0.01   0.004                 (66-165)     (66)    ______________________________________

Depending upon the the analyte of interest and the desired assayconfiguration, the preferred nonspecific binding blocker, as well as theoptimization of its concentration and whether it is included as acomponent of another assay reagent, is selected by empirical techniqueswhich can be performed without undue experimentation by one of ordinaryskill in the art of binding assays. In only one known instance, i.e.,the use of 0.005% dextran sulfate in the capture reagent of acompetitive digoxin assay, was there an inhibition of the bindingbetween the capture reagent and solid phase due to the addition of thenonspecific binding blocker.

Ion-capture Assay Devices

As described above, ion-capture assay devices may include impermeablesolid phase materials such as glass slides, magnetic particles, testtubes and plastic wells. However, it has also been discovered that theentire ion-capture assay can be performed in a porous solid phasematerial. The ion-capture assay devices of the present inventionspecifically involve any suitably absorbent, adsorbent, imbibing,bibulous, non-bibulous, isotropic or capillary possessing material(i.e., porous materials) through which a solution or fluid containingthe analyte can pass. The solution can be pulled or pushed through theporous material by suction, hydraulic, pneumatic, hygroscopic,gravitational or capillary forces, or by a combination thereof.

Possible assay devices include, but are not limited to, a conventionalchromatographic column, an elongated strip of porous material whereinthe fluid flow is substantially linear, a sheet wherein the fluid flowis linear or radial, a pad of porous material or a device involvingmultiple layered sheets or pads. The novel devices of the presentinvention involve the production of teststrips as well as flow throughdevices. For purposes of brevity, however, the following descriptionswill focus on teststrip devices, although the description of device zonecan be applied to both strip-type or layered flow through-type devices.Those skilled-in-the-art will readily appreciate the applicability ofthe present invention to flow through device formats.

One advantageously used solid phase porous material for the productionof a teststrip is nitrocellulose. Especially when a membranous solidphase material is used, the test sample and indicator reagent may bemixed prior to initiating fluid flow through the solid phase to obtain acontrolled, reproducible binding reaction between the analyte and theindicator reagent. Alternatively, the test device can further include apremixing application pad which is in fluid flow contact with theelongated strip and which optionally contains the indicator reagent. Thematerial of the application pad should be chosen for its ability topremix the test sample with the indicator reagent. For example, ifnitrocellulose is used as the solid phase, then a hydrophilicpolyethylene material or glass fiber filter paper are suitableapplication pad materials. Alternatively, if a solid phase material suchas glass fiber filter paper is used, then the indicator reagent can bereversibly immobilized on the elongated strip itself, either at thesample application site or at another site downstream from theapplication site. In yet other alternative devices and methods, theindicator reagent can be added to the device as a separate reagentsolution, either sequentially or simultaneously with the test sampleand/or capture reagent.

In alternative embodiments, a teststrip or flow-through device may bemade of a continuous piece of porous material containing diffusive orimmobilized reagents to form the various reagent and detection zones. Inyet another embodiment, a teststrip can be made from more than one solidphase material such that the different materials are in fluid flowcontact to allow the analyte to migrate from one material to another.The different materials may contain different diffusive or immobilizedassay reagents, with the individual material being assembled into anelongated strip or flow through pad device. In yet a further embodiment,two or more zones of the device may overlap. For example, the sampleapplication zone may also contain a diffusive assay reagent (e.g.,indicator reagent, capture reagent, etc.) which reacts with the analyteto form a complex or reactive product which continues to migrate toother zones in or on the device. In a further example, the sampleapplication zone may contain an immobilized assay reagent (e.g., polymeroppositely charged with respect to the capture reagent) whichimmobilizes the capture reagent or capture reagent complexes fordetection. Again, those skilled-in-the-art will readily appreciate theapplicability of the present invention to a variety of device formatswherein the indicator reagent is immobilized by directly or indirectlybinding to a capture reagent conjugate that is in turn immobilized by anoppositely charged solid phase material.

a. Application pad

If an application pad is used in a teststrip device, it is placed influid flow contact with one end of the porous material, referred to asthe proximal end, such that the test sample or an eluting solvent canpass or migrate from the application pad to the porous material. Fluidflow contact can include physical contact of the application pad to theporous material as well as the separation of the pad from the porousmaterial by an intervening space or additional material which stillallows fluid flow between the pad and the strip. Substantially all ofthe application pad may overlap the porous material to enable the testsample to pass through substantially any part of the application pad tothe proximal end of the elongated strip. Alternatively, only a portionof the application pad might overlap the elongated strip material. Theapplication pad can be any material which can transfer the test sampleand/or eluting solvent to the elongated strip and which can absorb avolume of test sample and/or eluting solvent that is equal to or greaterthan the total volume capacity of the elongated strip.

Materials preferred for use in the application pad includenitrocellulose, porous polyethylene pads and glass fiber filter paper.The material must also be chosen for its compatibility with the analyteand assay reagents, for example, glass fiber filter paper was found tobe the preferred application pad material for use in a human chorionicgonadotropin (hCG) assay device.

In addition, the application pad may contain one or more assay reagentseither diffusively or non-diffusively attached thereto. Reagents whichcan be contained in the application pad include, but are not limited to,indicator reagents, ancillary specific binding members, test samplepretreatment reagents and signal producing system components. Forexample, in a preferred embodiment of an ion-capture device an indicatorreagent is predeposited in the application pad during manufacture; thiseliminates the need to combine test sample and indicator reagent priorto using the device. The isolation of assay reagents in the applicationpad also keeps interactive reagents separate and facilitates themanufacturing process. For example, the indicator reagent may beretained in the application pad in a dry state, and upon contact withthe test sample or eluting solvent the indicator reagent isreconstituted and dispersed, thereby allowing its migration through thedevice. In yet another embodiment, the diffusive indicator reagent issituated on the teststrip material itself at a position between theapplication pad and a detection zone on the teststrip. In anotherembodiment, the diffusive indicator reagent is situated on the porousteststrip material at a detection zone, and that indicator reagent whichdoes not become immobilized at the detection zone due to the assayreaction will pass from the detection zone.

In a preferred ion-capture device, the application pad receives the testsample, and the wetting of the application pad by the test sample willperform at least two functions. First, it will dissolve or reconstitutea predetermined amount of reagent contained by the pad. Secondly, itwill initiate the transfer of both the test sample and the freshlydissolved reagent to the porous material. The application pad may servea third function as both an initial mixing site and a reaction site forthe test sample and assay reagent.

In another preferred embodiment, the application pad contains both theindicator reagent and the capture reagent in a dried form. The additionof the test sample reconstitutes the assay reagents, thereby enablingtheir reaction with the analyte and the formation of a charged indicatorreagent/analyte/capture reagent complex. The complex then migrates fromthe application pad to the porous teststrip material for subsequentreaction with a polymeric material immobilized in a detection zone,wherein that polymeric material is oppositely charged with respect tothe capture reagent. Alternatively, either the indicator reagent or thecapture reagent may be contained in the porous teststrip materialbetween the application pad and the detection zone. Preferably, thecapture reagent complex is allowed to form prior to or concurrent withthe migration of the capture reagent into the detection zone.

In another embodiment of the present invention, gelatin is used toencompass all or part of the application pad. Typically, suchencapsulation is produced by overcoating the application pad withgelatin. The effect of this overcoating is to increase the stability ofthe reagent contained by the application pad. The addition of testsample to the overcoated application pad causes the gelatin to dissolve,thereby rehydrating the predeposited assay reagent. In an alternativeembodiment of the present invention, the reagent containing applicationpad is dried or lyophilized to increase the shelf-life of the device.Lyophilized application pads were found to produce stronger signals thanair dried application pads, and the lyophilized application padsmaintained stability for longer periods.

In another preferred embodiment, the assay devices of the presentinvention can be further modified by the addition of a filtration means.The filtration means can be a separate material placed above theapplication pad or between the application pad and the porous material.Alternatively, the application pad material can be chosen for itsfiltration capabilities. The filtration means can include any filter ortrapping device used to remove particles or cells above a certain sizefrom the test sample. For example, the filter means can be used toremove red blood cells from a sample of whole blood, such that plasma istransferred to the porous material. Such filter means are disclosed byU.S. Pat. No. 4,477,575 which is hereby incorporated by reference.Optionally, the filter means can include a reagent or reagents to removeparticles or interferents from the test sample.

Another modification of the present invention involves the use of one ormore additional layers of porous material placed between the applicationpad and the porous material or overlayed upon the application pad. Suchan additional pad or layer can serve as a means to control the rate offlow of the test sample to or from the application pad. Such flowregulation is preferred when an extended incubation period is desiredfor the reaction of the test sample and the reagent(s) in theapplication pad. Alternatively, such a layer can contain an additionalassay reagent(s) which is preferably isolated from the application padreagents until the test sample is added. The flow control layer may alsoserve to prevent unreacted assay reagents from passing to the porousmaterial.

b. Porous Teststrip Material

The porous material used in the novel ion-capture devices of the presentinvention may be any suitably absorbant, porous or capillary possessingmaterial through which a solution containing the analyte can betransported by a wicking action. One preferred porous material forteststrip devices is nitrocellulose. When nitrocellulose is used,however, the material of the optional application pad should be chosenfor its ability to premix the test sample and one or more assayreagents: fluid flow through a nitrocellulose membrane is laminar anddoes not provide the more turbulent flow characteristics which allow theinitial mixing of test sample and application pad reagents within theporous material. If nitrocellulose is used as the porous material, thenPorex® hydrophilic polyethylene material or glass fiber filter paper areappropriately used as application pads to enable the mixing and reactionof the test sample and assay reagents within the application pad. Anespecially preferred porous material is glass fiber filter paper.

The particular dimensions of the porous strip material will be a matterof convenience, depending upon the size of the test sample involved, theassay protocol, the means for detecting and measuring the signal, andthe like. For example, the dimensions may be chosen to regulate the rateof fluid migration, as well as the amount of test sample to be imbibedby the porous material and transported to or through the detection site.

As discussed above, in a binding assay the detection site is typicallyformed by directly or indirectly attaching a charged polymer to theporous material at a predetermined location. Direct attachment methodsinclude adsorption, absorption and covalent binding. Indirect attachmentmethods include the use of insoluble microparticles, to which thecharged reagent has been attached, wherein the particles are retainedand immobilized in or on the porous support material. The means ofattaching a reagent to the microparticles encompasses both covalent andnon-covalent means, that is adhered, absorbed or adsorbed. It ispreferred that ion-capture reagents be attached to the microparticles bycovalent means.

It is also within the scope of this invention to attach more than onereagent to the microparticles which are then immobilized within theporous material. For example, to slow or prevent the diffusion of thedetectable reaction product in an enzyme/substrate signal producingsystem, the substrate can be immobilized within the porous material. Thesubstrate can be immobilized by direct attachment to the porous materialby methods well-known in the art or the substrate may be immobilized bybeing covalently bound to insoluble microparticles which have beendeposited in and/or on the porous material.

The size of the particles may vary depending upon the type of porousmaterial used as well as the type of material from which the particle ismade. For example, in a glass fiber porous material, glass andpolystyrene particles should be of sufficient size to become entrappedor immobilized in the pores of the porous material and not move whenconfronted by the migrating fluid. In the same glass fiber matrix, muchsmaller latex particles can be used because the latex particlesunexpectedly affix themselves to the glass fibers by an unknownmechanism. Thus, unlike pore size dependent glass and plastic particles,the latex particles are pore size independent, and lot-to-lot variationsin pore size of the porous material will not adversely affect theperformance of the device. As a result, one particularly preferredbinding assay device uses latex particles, having capture reagentattached thereto, distributed in a glass fiber porous material. Thedistribution of the microparticles or other reagents onto or into thematrix of the porous material can be accomplished by reagent printingtechniques as are well-known to those skilled-in-the-art.

The ion-capture reagent, signal producing component or reagent-coatedmicroparticles can be deposited singly or in various combinations on orin the porous material. They can be deposited in a variety ofconfigurations to produce detection or measurement sites of varyingshape. For example, a reagent can be deposited as a discrete situshaving an area substantially smaller than that of the entire porousstrip material.

Alternatively, the reagent can be distributed over the entire porousmaterial in a substantially uniform manner to form a capture site ordetection site that substantially includes the entire porous material.In this instance, the extent of signal production along the length ofthe detection site, or the distance of the detectable signal from theproximal end of the porous material, is then related to the amount ofanalyte in the test sample. The amount of analyte can be determined bythe comparison of the length or distance of the resulting signal tothose observed for calibrated standards.

In another embodiment, the reagent can be distributed as a narrowstripe. Use of the narrow stripe, rather than a uniform distribution ofreagent, can serve to sharpen the image of the detectable signal on theporous material. Furthermore, more than one narrow parallel stripe canbe distributed along the length of the porous material, wherein thereagent within each stripe is directed to a different analyte, therebyforming a multi-analyte assay device. As an addition to those devices inwhich the length or distance of analyte travel is measured, a scale ofappropriate symbols, numbers or letters can be imprinted upon the porousmaterial to aid in the measurement and thus the quantitation of analyte.

In another embodiment, the reagent can be distributed more lightly atone end of the porous material than at the other. In a competitivebinding assay, this deposition of capture reagent in a gradient fashionprovides for greater sensitivity at the end of the porous materialhaving the lighter distribution, because of the more rapid displacementof the indicator reagent from the capture reagent binding sites by theanalyte.

In alternative embodiments, the appropriate capture and signal producingreagents can be distributed in any pattern convenient for detectionincluding, but not limited to, numerals, letters, dots and symbols suchas "±", "%" or the like which display the detectable signal uponcompletion of the assay. Reaction matrices can optionally be preparedwith the assay reagents incorporated into the material in an overlappingdesign, such that the reaction of one reagent completes one portion of adetectable pattern and a second reaction completes another portion ofthe detectable pattern. For example, one reaction may complete thevertical portion of a "cross" shaped design while a second reactioncompletes the horizontal portion of the cross. Alternatively, oneportion of the design may be visible or detectable prior to performanceof the assay, with a single reaction completing the overall design. Thecompletion of the vertical portion alone would typically indicate anegative assay result, whereas completion of both portions of thedetectable design would indicate a positive assay result. Any pattern ordesign may be used, however, wherein the partial formation of the designindicates other than a positive assay result and the complete formationof the design indicates a positive assay result. Such methods anddevices are described in U.S. Pat. No. 4,916,056 the disclosure of whichis hereby incorporated by reference.

In yet another embodiment, the reagents can be distributed as a seriesof parallel bars which traverse the width of the porous strip materialand which are spaced from about the proximal end of the porous materialto about the distal end, thereby creating a ladder-like capture situsconfiguration. As with the narrow-stripe configuration, the bars and theintervening spaces serve to sharpen the image of the signal produced onthe porous material. The number of bars at which signal is detectablecan be counted and correlated to the amount of analyte in the testsample. When the bars are spaced closely together, the device providesless analytical sensitivity but greater amounts of analyte can bemeasured. Alternatively, by spacing the bars further apart, increasinglygreater sensitivity can be obtained. It is also within the scope of thisinvention to vary the sensitivity within different portions of theporous material depending upon whether greater discriminationsensitivity for the analyte is required at the high end or low end ofits concentration range. Another variation of the parallel barconfiguration involves the use of multiple capture or reaction reagentswherein the reagents within the capture and detection sites are directedto a different analyte, thereby forming a multi-analyte assay device.

The particular dimensions of the solid phase will be a matter ofconvenience and will depend upon the size of the test sample involved,the assay protocol and the means for detecting and measuring the signal.For example, the dimensions may be chosen to regulate the rate of fluidmigration as well as the amount of test sample to be imbibed by thesolid phase.

Predetermined amounts of assay reagents can be incorporated within thedevice, thereby reducing or avoiding the need for additionalmanipulation by the user. Thus, it is within the scope of this inventionto incorporate more than one reagent within the device. For example, toslow or prevent the diffusion of the detectable reaction product in anenzyme/substrate signal producing system, the substrate can beimmobilized within the teststrip. The substrate can be immobilized on orin the teststrip by methods well-known in the art, or the substrate maybe immobilized by being covalently bound to insoluble microparticleswhich have been deposited in and/or on the teststrip. More than oneassay reagent may be present in any given reagent zone or site on thedevice so long as the reagents do not react until contacted with thetest sample or eluting solvent.

The various signal display formats or patterns described above can alsoincorporate assay controls to confirm the efficacy of the assayreagents, the completion of the assay or the proper performance of theassay. Such controls are well-known to those skilled-in-the-art. It isalso within the scope of this invention to have a reagent, at the distalend of the teststrip device, which indicates the completion of the assay(i.e., an end of assay indicator to signal that the test sample hascompleted its migration through the device). For example, the completionof the assay may be shown by a change of color at the control site uponcontact with the test solution, wicking solution or a signal producingcomponent. Reagents which would change color upon contact with anaqueous test solution include the dehydrated transition metal salts,such as CuSO₄, Co(NO₃)₂, and the like. The pH indicator dyes can also beselected to respond to the pH of the buffered wicking solution. Forexample, phenolphthalein changes from clear to intense pink upon contactwith a wicking solution having a pH range between 8.0-10.0.

A test sample can be contacted to the teststrip by applying the testsample to an application site or by immersing the application site inthe test sample. In a sheet-like device having radial capture andconjugate recovery sites, the sample is applied to a central applicationsite. Prior to contacting the sample to the solid phase, the sample canalso be mixed with additional reagents such as the indicator reagent,capture reagent, buffers or wicking reagents (i.e., reagents whichfacilitate the transport of the test sample through the solid phase). Ina further embodiment, the test sample can be applied to one portion ofthe teststrip, upstream of the capture site, with one or more of theadditional reagents being applied to yet another portion of theteststrip upstream of the test sample application site.

In yet another embodiment, the device can include an additionalabsorbent material positioned downstream from or beneath the capturesite. It will be appreciated that the absorbent material can serve toincrease the amount of test sample and indicator reagent which passesthrough the capture and detection sites on the solid phase.

When small quantities of non-aqueous or viscous test samples are appliedto the device, it may be necessary to employ a wicking solution,preferably a buffered wicking solution, to facilitate the migration ofthe assay reagent(s) and test sample through the device. When an aqueoustest sample is used, a wicking solution generally is not necessary butmay be used to improve flow characteristics or adjust the pH of the testsample. In immunoassays, the wicking solution typically has a pH rangefrom about 5.5 to about 10.5, and more preferably from about 6.5 toabout 9.5. The pH is selected to maintain a significant level of bindingaffinity between the specific binding members and the analyte. When thelabel component of the indicator reagent is an enzyme, however, the pHmust also be selected to maintain significant enzyme activity for colordevelopment in enzymatic signal production systems. Suitable buffersinclude, but are not limited to, phosphate, carbonate, barbital,diethylamine, tris(hydroxymethyl)-aminomethane (Tris),2-amino-2-methyl-l-propanol and the like. The wicking solution and thetest sample can be combined prior to contacting the test device, or theycan be contacted to the application pad separately.

c. Flow-through Assay Devices

Conventional flow-through devices have at least a substantially planarlayer including a sample-contacting surface wherein a nondiffusivespecific binding member is disposed for the immobilization of theanalyte of interest. The layer is positioned such that when the deviceis used in the performance of a binding assay, at least a portion of thetest sample that contacts the first surface passes through the firstsurface to an opposing second surface.

Typically, the flow-through devices include a second layer or absorbentmeans for absorbing fluid passing through first layer, wherein theabsorbent means is in direct contact with the second surface of thefirst layer, or is in close enough proximity that fluid passing throughthe second surface is transported to the absorbent means. In modifieddevices, the absorbent means may be spaced from the first layer and canbe contacted to the second surface of the first layer by subsequentlypressing the layers together.

Optionally, the flow-through devices may also involve a filtering meansdisposed in relation to the first layer such that when the device is inuse the test sample will pass through the filtering means prior tocontacting the first surface. Furthermore, flow-control means may bedisposed between the first layer and the absorbent means to adjust therate of flow of fluids from the first layer. Back-flow control means mayalso be disposed between the first layer and the absorbent means toprevent the migration of signal producing substances from the absorbentmeans to the first layer.

The flow-through devices may also include an assay reagent layer orlayers disposed in relation to the first layer, such that when thedevice is in use, sample fluid passes through the assay reagent layerprior to contacting the first surface. The assay reagent is typicallyresolubilized by the addition of test sample to the reagent layer andthe reagent is then available for further reaction with the analyte orother reagents housed within the assay device. Other embodiments mayinclude a filter layer or a combination filter/reagent layer. Stillother devices may involve a removable filter and/or reagent layer.

The novel flow-through assay devices of the present invention, involve acontact surface wherein a charged polymer is disposed for thenonspecific binding and immobilization of the oppositely charged capturereagent and complexes thereof. The device may consist of a layer or afirst layer in combination with one or more other device layersdescribed above. For example, one or more pre-reaction layers maycontain the indicator reagent and or the capture reagent such that theanalyte is allowed to contact the assay reagents prior to contacting theion-capture surface of the flow-through device.

In either the flow-through or teststrip assay devices, one or more assayreagents, such as the indicator reagent or capture reagent, may beapplied to the device during the performance of the assay. The preferredembodiments of the present invention, however, involve the incorporationof all necessary assay reagents into the assay device so that only atest sample, and in some instances a wicking solution or elutingsolvent, need be applied to the device.

The present invention further provides kits for carrying out bindingassays. For example, a kit according to the present invention cancomprise the assay device with its incorporated reagents, and canoptionally include a wicking solution and/or test sample pretreatmentreagent as described above which are not incorporated in or on thedevice. Other assay components known to those skilled-in-the-art, suchas buffers, stabilizers, detergents, non-specific binding inhibitors,bacteria inhibiting agents and the like can also be present in the assaydevice and wicking solution.

EXAMPLES

The following Examples illustrate preferred ways of making the novelmaterials of the present invention and performing assay procedures usingthose materials. The Examples, however, are intended only to beillustrative, and are not to be construed as placing limitations uponthe scope of the invention, which scope is defined solely by the claims.

Example 1 Sandwich Assay for Carcinoembryonic Antigen (CEA)

a. Preparation of an anti-CEA antibody-PGA capture reagent

The following sequence of steps describes the chemistry employed for thepreparation of an antibody/polyglutamic acid (PGA) conjugate, i.e., anantibody/anionic polymer capture reagent.

Preparation of a traceable anionic polymer: The sodium salt of PGA (onegram; 7.14×10⁻⁵ mole; average molecular weight MW! 14,000; Sigma) wasconverted to 3-(2-pyridyl-dithio) propionyl-PGA (PDP-PGA) by the methodof Tsukada, et al. (JNCI; 73; 721-729, 1984) with the followingprocedural modifications. The PDP-PGA was not reduced to the freesulfhydryl prior to the thiopropyl sepharose 6B isolation. Instead, thePDP-PGA was dissolved in 0.1M Na phosphate and 1 mM EDTA (pH 6.5) andstirred with thiopropyl sepharose 6 B (60 ml; 30 grams; PharmaciaChemicals, Uppsala, Sweden). After dialysis and lyophilization, a 24%yield of the PDP-PGA conjugate was obtained (0.244 grams; 1.72×10⁻⁵mole).

To ensure that the disulfide was maintained during the ensuingchemistries, the thiopyridyl group was exchanged for a5-thio-2-nitrobenzoate (TNB) protecting group. A 100 mole excess of1,4-dithiothreitol (MW 154.2) was added to a solution of the PDP-PGA (20mg; 1.42×10⁻⁶ mole) dissolved in 0.1M sodium phosphate (4.0 ml; pH 7),and the reaction was run for one hour at 40° C. The mixture was dilutedto ten milliliters with 5.0 mM sodium acetate, 0.14M NaCl, and 1.0 mMEDTA (pH 5.5) and dialyzed in 2000 molecular weight cut off (MWCO)tubing against the dilution buffer. Dialysis was continued againstdistilled water, followed by lyophilization. The yield of thiopropyl-PGA(HS-PGA) was 13.5 mg. The HS-PGA (13.5 mg) was dissolved in 0.1M sodiumphosphate (pH 7.0; 9.6×10⁻⁷ mole) and reacted with a 10 mole excess of5,5' dithiobis (2-nitrobenzoic acid) (DTNB) for one hour at roomtemperature. This mixture was diluted to ten milliliters with 0.1Msodium phosphate (pH 7) and dialyzed in 2000 MWCO tubing against thedilution buffer. Dialysis was continued against distilled water and wasfollowed by lyophilization to produce 5-(2-nitrobenzoic dithio)propionyl-PGA (TNB-PGA; 8.5 mg; 6.07×10⁻⁷ mole).

To trace the number of anionic polymer molecules attached to eachcapture reagent antibody, the TNB-protected PGA was then labeled with anethylenediamine derivative of fluorescein. The TNB-PGA was loaded withan ethylenediamine derivatized fluorescein (EDA-FI; MW 532) bydissolving TNB-PGA (8.5 mg) in dry N-N dimethyl-formamide (2.0 ml),treating with a 90 mole excess of N-methylmorpholine (MW 101.15),lowering the temperature to 0° C., and adding a 90 mole excess ofisobutylchloroformate (MW 136.58). This reaction was run at 0° C. forone hour. The mixture was warmed to room temperature, a 30 mole excessof EDA-FI was added, and the reaction was run at room temperature withstirring overnight. The mixture was diluted to ten milliliters with 0.1Msodium phosphate (pH 7.0) and dialyzed in 2000 MWCO tubing against thedilution buffer. Dialysis was continued against distilled water and wasfollowed by lyophilization to yield TNB-PGA/EDA-FI conjugate (7.8 mg;5.6×10⁻⁷ mole).

The TNB group was removed by dissolving the TNB-PGA/EDA-FI (7.8 mg) in0.1M sodium phosphate (3.0 ml; pH 7.0) and treating with 100 mole excessof 1,4-dithiothreitol for one hour at 40° C. The reaction was monitoredfor a shift of a 334 nm to a 412 nm peak on a UV/VIS spectrophotometer.The material was diluted to ten milliliters with distilled water anddialyzed in 2000 MWCO tubing against distilled water. Uponlyophilization, thiopropyl-PGA/EDA-FI (HS-PGA/EDA-FI; 8.4 mg) wasobtained. At this point, a UVNIS scan was taken to determine the numberof fluoresceins per PGA molecule (i.e., loading). A value of 0.81fluoresceins per PGA was calculated for this preparation.

Antibody activation: The monoclonal antibody, an anti-CEA antibody wasmaleimide activated per the method of Tuskada, et al. (JNCI: 73;721-729, 1984) with the following exceptions. The antibody concentrationwas one mg/ml, and a 150 mole excess of N-succinimidyl m-(N-maleimido)benzoate (SMBE, MW 314.3; Sigma) was used. It was determinedexperimentally that a 150 mole excess was necessary to introduce betweenthree and five maleimide groups to the anti-CEA antibody. Clean-up wasperformed using the Meares, et al. centrifuge method (AnalyticalBiochemistry: 1142; 68-78, 1984) with Sephadex G-50/80 (Sigma) in threemilliliter syringe columns. The number of maleimides per antibody wasdetermined using the titration method of Liu, et al., (Biochemistry: 18;690-696, 1979). It was found that 4.6 maleimides were introduced perantibody during this antibody activation.

The thiopropyl-fluorescein-labeled PGA was then reacted with themaleimide derived antibody to yield the antibody/PGA conjugateappropriate for a carcinoembryonic antigen ion-capture immunoassay. Themaleimide-activated antibody (1.0 mg; 6.25×10⁻⁹ mole) in 0.1M sodiumphosphate (1.0 to 2.0 ml; pH 7.0) was pH adjusted to 6.5 with 1.0N HCl.Then, a 10 mole excess of HS-PGA/EDA-FI (approximately 1.0 mg) in 0.1Msodium phosphate (100 μl ) was added to the activated antibodypreparation. The conjugation was run overnight with gentle stirring atroom temperature. The mixture was diluted to ten milliliters in 0.1Msodium phosphate (pH 7.0) and dialyzed in 50,000 MWCO tubing against0.001M Na phosphate (pH 7.0) followed by lyophilization. The drymaterial was redissolved in distilled water (0.25 ml) and highperformance liquid chromatography (HPLC) fractionated for the largestpeak at A280. The chromatography was performed using a Bio-Sil TSK250(Bio-Rad Laboratories, Richmond, Calif.) 300 mm×7.5 mm column, eluted atone milliliter/minute with 50 mM sodium sulfate, 20 mM sodium phosphate,and 0.3M NaCl (pH 6.8).

The largest peak was assayed for protein content using Bio-Rad'sBradford assay with a bovine IgG standard. The peak contained 95.5 μg/mlprotein equating to 5.97×10⁻⁷ molar protein (IgG MW 160,000). Byscanning the UV/VIS and taking the absorbance at 494 nm, it wasdetermined that this fraction also contained 2.12×10⁻⁶ molarfluorescein, corresponding to 3.6 fluoresceins per antibody molecule.Knowing that there were 0.81 fluoresceins per PGA molecule, this equatedto 4.4 PGA molecules conjugated to each antibody. The peak fraction wasfrozen and subsequently used in the assay.

An important aspect of the above described chemistries is that thereexists but a single site of attachment between each polymeric anion andthe antibody. A solitary covalent link between the two circumvents thepotential intermolecular and intramolecular crosslinking that couldoccur if a polymeric anion having multiple activated groups wereemployed.

As an alternative to the above capture reagent example, a cationicderived antibody could also be formed for use in conjunction with ananionic solid phase material.

b. Preparation of the solid phase

The solid phase fibrous matrix of a disposable flow-through material wascoated with a polymeric quaternary compound to give the solid phase apositive charge. Celquat® L-200 polymeric compound, a water solublecellulose derivative, was used. A 1% aqueous solution of Celquat® L-200polymeric compound (50 μl) was applied to the solid phase material,followed by a wash of diluent containing 300 mM NaCl, 50 mM Tris and0.1% NaN₃ (75 μl; pH 7.5).

c. Preparation of the indicator reagent

The indicator reagent consisted of a conjugate of alkaline phosphataseand anti-CEA antibody fragment, which binds to a different epitope thanthe antibody specified in the capture reagent. The alkalinephosphatase-labeled anti-CEA antibody fragment was in a buffercontaining: 50 mM Tris, 50 mM NaCl, 1.0 mM MgCl₂, 0.1 mM ZnCl₂, 5.0 mMsodium tartrate, 0.5% calf skin gelatin, and 3% mouse serum.

d. Immunoassay protocol-determination of CEA

The indicator reagent (70 μl) was placed into a reaction well. Then,buffered capture reagent (20 μl of anti-CEA/PGA conjugate in a buffer of50 mM Na₂ SO₄, 20 mM sodium phosphate, and 300 mM NaCl at pH 6.8) wasadded to the well. A 35 μl specimen containing CEA was added to thewell, and the homogeneous immunoreaction mixture was incubated for 20minutes at 34.5° C. Four different specimens were run in the assay, eachof which was a CEA calibrator from the Abbott Laboratories CEA enzymeimmunoassay kit. An aliquot of each reaction mixture (100 μl) was thenapplied to the solid phase material, followed by three 75 μl washes ofdiluent. Finally, an enzyme substrate (70 μl; 1.2 mM4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 1.0 mMMgCl₂, 0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3) was added at 34.5°C. for reaction with the indicator reagent, and the resulting rate offluorescence was measured. The dose-response results of the assay areshown in Table 1. The results demonstrate that as the CEA test sampleconcentration increased there was a corresponding increase in theformation of capture reagent/analyte/indicator reagent complex, andtherefore, the amount of detectable label associated with the solidphase increased.

                  TABLE 1    ______________________________________    CEA Ion-capture Sandwich Assay    Capture reagent: anti-CEA antibody-PGA conjugate    Indicator reagent: alkaline phosphatase-labeled anti-CEA antibody    fragment    CEA (ng/ml)  Rate (counts/sec/sec)    ______________________________________    0            37    4            170    30           931    80           2398    ______________________________________

Example 2 Competitive Inhibition Assay of Mouse Immunoglobulin

a. Preparation of an IgG-PGA capture reagent

A protein-A affinity purified mouse monoclonal immunoglobulin G wascoupled to negatively charged PGA using a water-soluble carbodiimidereagent (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide; EDCI)according to the following procedures.

Fluorescein-labled PGA (10 mg; FI-PGA) was added to an ice-cold solutionof the antibody (4.8 mg/ml) in phosphate-buffered saline (PBS; 75 mM KH₂PO₄ and 300 mM NaCl at ph 7.2). To that solution was added a freshlyprepared ice-cold solution of EDCI (100 μl; 10 mg/ml), and the resultantreaction mixture was allowed to warm to room temperature with continuesstirring for 2.5 hours. An additional freshly prepared ice-cold solutionof EDCI (50 μl; 100 mg/ml) was then added to the reaction mixture withrapid stirring. The reaction mixture was stirred for another 1.5 hours.The mixture was then fractionated by gel filtration chromatography usinga Spherogel TSK-3000SWG column (2.15 cm×30 cm) fitted with a SpherogelTSK-G guard column (2.15 cm×7.5 cm; Beckman Instruments, Inc.,Fullerton, Calif., 92634). The column was eluted with PBS at a flow rateof five milliliters/minute. The PGA/antibody ratio of these pools wasdetermined by quantitating the fluorescence in the FI-PGA conjugates ofthe antibody. The results are shown in Table 2.

                  TABLE 2    ______________________________________    Mouse IgG-PGA conjugates prepared using EDCI    Pool      Peak Molecular Weight                             PGA/antibody    ______________________________________    I         420,000        3.8    II        280,000        4.1    III       220,000        5.5    ______________________________________

b. Preparation of the solid phase

A porous fibrous matrix material was coated with a polymeric quaternaryammonium compound (GAFQuat™ 755N quaternary ammonium compound; GAFCorporation) to form the solid phase. An aqueous solution of 0.5%GAFQuat™ quaternary ammonium compound (50 μl) was applied to the surfaceof the material, followed by a water wash (75 μl).

c. Binding of the indicator reagent to the capture reagent

The indicator reagent, an alkaline phosphatase conjugate of sheepanti-mouse immunoglobulin (Jackson ImmunoResearch Laboratories, Inc.;West Grove, Pa., 19390), was diluted in Tris-buffered saline containing1% fish gelatin 25 mM Tris (hydroxymethyl) aminomethane and 100 mM NaCl,pH 7.5!. The capture reagent of PGA/mouse monoclonal antibody conjugate(Pool I of Table 2) was similarly treated. Two hundred microliters ofeach reagent was added to a series of test tubes which were thenincubated at 37° C. for 30 minutes. An aliquot of the reaction mixture(75 μl) was applied to the solid phase, immediately followed by three150 μl washes of Tris-buffered saline. Finally, an enzyme substrate (70μl of 1.2 mM 4-methylumbelliferylphosphate in a solution of 100 mM AMP,1 mM MgCl₂, 0.1% NaN₃, and 4 mM tetramisole; pH 10.3) was added to thematerials at 32.7° C., and the resulting rate of fluorescence wasmeasured. The results of the experiment are summarized in Tables 3 and4.

                  TABLE 3    ______________________________________    Dose response of capture reagent/indicator reagent binding    PGA/antibody* (μg/ml)                   Rate of fluorescence (counts/sec/sec)    ______________________________________    10             1559    1              816    0.1            179    0.01           70    0              36    ______________________________________     *The initial concentrations of PGAcoupled-antibody before mixing with a     1000fold diluted alkaline phosphataselabeled sheep antimouse     immunoglobulin.

                  TABLE 4    ______________________________________    Dose response of indicator reagent/capture reagent* binding    Indicator reagent titer**                   Rate of fluorescence (counts/sec/sec)    ______________________________________    10.sup.2       5062    10.sup.3       796    10.sup.4       93    10.sup.5       10    10.sup.6       5    ______________________________________     *The initial concentration of PGAcoupled-antibody before mixing with     alkaline phosphataselabeled sheep antimouse immunoglobulin was five     μg/ml.     **The indicator reagent titer is the reciprocal of the dilution of the     reagent stock.

d. Competitive inhibition assay for mouse IgG

The capture reagent and indicator reagent were prepared as describedabove. All of the reagents were diluted in Tris-buffered salinecontaining 1% fish gelatin. The indicator reagent was diluted 1000-foldfrom the stock solution, and the capture reagent was diluted to tenμg/ml. In a series of test tubes, 150 μl each of appropriately dilutedindicator reagent, capture reagent, and mouse monoclonal antibody weremixed. The mixtures were incubated at 37° C. for 30 minutes. Aliquots ofthe mixtures (75 μl) were applied to the solid phase, immediatelyfollowed by three 150 μl washes of Tris-buffered saline. An enzymesubstrate (70 μl of 1.2 mM 4-methylumbelliferylphosphate in a solutionof 100 mM AMP, 1 mM MgCl₂, 0.1% NaN₃, and 4.0 mM tetramisole; pH 10.3)was then added to the solid phase at 32.7° C., and the resulting rate offluorescence was measured. The results of this example illustrating acompetitive inhibition assay for mouse IgG are shown in Table 5. Theresults demonstrate that as the mouse monoclonal antibody concentrationincreased there was a corresponding decrease in the formation of capturereagent/indicator reagent complex, and therefore, the amount ofdetectable label associated with the solid phase decreased.

                  TABLE 5    ______________________________________    Inhibition of indicator reagent binding due to mouse monoclonal    antibody    Capture reagent: PGA/mouse monoclonal IgG conjugate    Indicator reagent: alkaline phosphatase-sheep anti-mouse    immunoglobulin conjugate    Mouse IgG (μg/ml)                 Rate of fluorescence (counts/sec/sec)    ______________________________________    0            110    3.3 × 10.sup.-3                 106    3.3 × 10.sup.-2                 98    3.3 × 10.sup.-1                 67    3.3          36    33           10    ______________________________________

Example 3 Sandwich Assay for Human Chorionic Gonadotropin (hCG)

a. Preparation of the capture reagent

A highly negatively charged albumin derivative was prepared and coupledto anti-hCG antibodies to form the capture reagent according to thefollowing procedures.

Modification of rabbit serum albumin to form a negatively chargedprotein derivative: Rabbit serum albumin (RSA) was extensivelysuccinylated and coupled with para-azobenzenesulfonate by the procedureof Jou, et al., (Methods in Enzymology: Vol. 92, Part E; 257-276,Academic Press, 1983). Two per cent RSA in phosphate-buffered saline(PBS, 14 ml, pH 8.0) was mixed with 5% succinic annhydride inpara-dioxane (2.28 ml). The pH was maintained at 8 by the addition of1.0N NaOH. The reaction mixture was stirred at room temperature for 30minutes. Hydroxylamine hydrochloride was added (0.6 g) and the pH of thesolution was adjusted to 9.5 by adding an appropriate amount of 5N NaOH.The mixture was then dialyzed against water. The resultant SUC₆₅ -RSAwas coupled to para-azobenzenesulfonate according to the followingreactions.

A suspension of para-azobenzenesulfonic acid (0.15 mmole, 26 mg) in 1NHCl (0.8 ml) was cooled in an ice bath and treated with 1N NaNO₂ (0.2ml) for 30 minutes with rapid stirring. The resultant diazonium saltsolution was added by drops to the ice cooled SUC₆₅ -RSA solution withrapid stirring. The pH of the reaction mixture was maintained at 11 bythe addition of 1.0N NaOH. The dark red reaction mixture was stirred andallowed to warm to room temperature for one hour before it wasextensively dialyzed against water. The resultant Sp-SUC₆₅ -RSA anionicderivatized protein was kept refrigerated until used.

Preparation of anti-hCG F(ab')₂ fragments: Anti-hCG F(ab')₂ fragmentswere prepared according to the method of Nisonoff, et al., (Arch.Biochem. Biophy.: 89; 230-244, 1960) from affinity purified goatanti-hCG antibodies. A portion of affinity purified antibody solution inphosphate buffered saline (pH 7.2) was acidified to pH 4 by addingacetic acid. The preferred concentration of antibodies at this point wasone mg/ml. Pepsin was added to reach a final concentration of 20 μg/ml.The mixture was incubated at 37° C. overnight. The reaction was stoppedby adding 6.0N NaOH to bring the reaction mixture to a pH of 7.5. Thedigested antibody fragments solution was concentrated to 20 mg/ml. TheF(ab')₂ fragments were purified by gel-filtration high performanceliquid chromatography using a Spherogel TSK-3000SWG column (2.15 cm×30cm) fitted with a Spherogel TSK-G guard column (2.15 cm×7.5 cm).

Preparation of anti-hCG TNB-Fab' fragments: Anti-hCG Fab' fragments wereprepared and derivatized into a thiol-reactive form according to amodification of the methods of Parham, et al., (J. Immunol. Method.: 53:133-173, 1982) and Brennan, et al., (Science: 229: 81-83, 1985). Withstirring, a solution (158 μl) of 0.1M NaAsO₂ containing 20 mM EDTA wasadded to 1.28 ml of goat F(ab')₂ (goat anti-human chorionic gonadotropinantibody fragment, 16 mg/ml) containing trace ¹²⁵ I-F(ab')₂ in PBS. Thereductive cleavage reaction was started by adding 0.1M cysteine-HCl (158μl). The reaction mixture was overlayed with nitrogen and incubated withstirring at 37° C. for one hour. The reaction was then quenched byadding 19 mg of 5,5'-dithiobis-(2-nitrobenzoic acid). After stirringovernight at room temperature, the mixture was chromatographed on aPD-10 column (Pharmacia Inc., Piscataway, N.J.) preequilibrated withPBS, and then chromatographed on a size exclusion high performanceliquid chromatography column Spherogel TSK-2000SWG column (2.15 cm×30cm) fitted with a Spherogel TSK-G guard column (2.15 cm×7.5 cm)!. Thepurified thionitrobenzoate derivative of Fab' (TNB-Fab') wasconcentrated to 7.9 mg/ml using a CX-10 ultrafiltration unit (MilliporeCorp., Bedford, Mass.).

Coupling of anti-hCG TNB-Fab' fragments to Sp-SUC₆₅ -RSA: A solution of1M dithiothreitol (DTT; 86 μl) was added to a solution (4.2 ml)containing Sp-SUC₆₅ -RSA (2.2 mg/ml) in 37.5 mM sodium phosphate, 150 mMNaCl, and 2.0 mM EDTA (pH 6.8). The mixture was incubated at 37° C. forthree hours and then at room temperature overnight. The resultingreaction mixture was chromatographed on a 2.5 cm×20 cm column packedwith Sephadex™ G-25 (Pharmacia Inc.) and preequilibrated with 75 mMsodium phosphate, 300 mM NaCl, and 2.0 mM EDTA (pH 6.8). A twomilliliter portion of the pooled fractions of reduced Sp-SUC₆₅ -RSA(0.48 mg/ml) was mixed with anti-hCG TNB-Fab' (0.15 ml; 7.9 mg/ml). Themixture was stirred at room temperature overnight. The reaction mixturewas then treated with 100 mM iodoacetic acid (107 μl) and stirred forone hour at room temperature. The Fab'-Sp-SUC₆₅ -RSA conjugate waspurified by size exclusion high performance liquid chromatography usinga Spherogel TSK-3000SWG column (2.15 cm×30 cm) fitted with a SpherogelTSK-G guard column (2.15 cm×7.5 cm).

Coupling of anti-hCG antibodies to Sp-SUC₆₅ -RSA: A solution (27 μl) of30 mM succinimidyl 4-(N-maleimido-methyl)-cyclohexane-1-carboxylate inN,N-dimethylformamide was added to 2.25 ml of affinity purified goatanti-hCG antibody (3 mg/ml) in PBS. The resulting reaction mixture wasstirred for one hour at room temperature and then chromatographed on aPD-10 column preequilibrated with 75 mM sodium phosphate, 300 mM NaCl,and 2.0 mM EDTA (pH 6.8). A 1.8 ml portion of the pooled fractions ofmodified antibodies (1.6 mg/ml) was mixed with three milliliters of theDTT-reduced Sp-SUC₆₅ -RSA (0.48 mg/ml). After stirring at roomtemperature overnight, the reaction was quenched by adding 100 mMiodoacetic acid (0.25 ml) and stirring at room temperature for one hour.The antibody-Sp-SUC₆₅ -RSA conjugate was purified by size exclusion highperformance liquid chromatography in the manner described above.

b. Preparation of the indicator reagent

The indicator reagent consisted of an alkaline phosphatase-goat anti-hCGantibody conjugate (prepared by coupling anti-hCG antibody to periodateactivated alkaline phosphatase) in an assay buffer containing 25 mM Tris(hydroxymethyl) aminomethane, 100 mM NaCl, 1 mM MgCl₂, 0.1 mM ZnCl₂,0.07% NaN₃, and 1% fish gelatin at pH 7.5.

c. Sandwich immunoassay protocol for hCG

The ion-capture immunoassay protocol included the use of a solid phaseprepared substantially in accordance with the method described inExample 2, the indicator reagent (alkaline phosphatase-goat anti-hCGantibody conjugate), one of two different capture reagents (goatanti-hCG Fab'-Sp-SUC₆₅ -RSA and goat anti-hCG IgG-Sp-SUC₆₅ -RSA) asprepared in Example 3.a. above, and a purified hCG standard solution.All reagents were appropriately diluted (as determined by a titer curve)in the assay buffer. Equal volumes (750 μl) of the indicator reagent andhCG sample solution were placed in a series of test tubes. Afterincubation at 37° C. for 30 minutes, a 125 μl aliquot of each incubatedmixture was mixed in a separate tube with an equal volume of a capturereagent. The resulting mixtures were incubated for 30 minutes. The assaymixture (75 μl) was then added to each solid phase material. The solidphase materials were then washed three times with 150 μl amounts ofwashing buffer 25 mM Tris (hydroxymethyl) aminomethane, 100 mM NaCl, 1.0mM MgCl₂, 0.1 mM ZnCl₂, and 0.07% NaN₃ at pH 7.5!. An enzyme substrate(70 μl of 1.2 mM 4-methylumbelliferylphosphate in a solution of 100 mMAMP, 1.0 mM MgCl₂, 0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3) wasthen added to the solid phase materials. The resulting rate offluorescence was measured at 32.7° C. The results of the experiment aresummarized in Table 6. The results demonstrate that as the hCG testsample concentration increased there was a corresponding increase in theformation of capture reagent/analyte/indicator reagent complex, andtherefore, the amount of detectable label associated with the solidphase increased.

                  TABLE 6    ______________________________________    hCG Ion-capture Sandwich Assay Comparing Different Capture    Reagents    Indicator reagent: hCG-specific goat IgG-alkaline phosphatase           Rate of fluorescence (counts/sec/sec)           hCG-specific capture reagents    hCG (mlU/ml)             Goat IgG-Sp-SUC.sub.65 -RSA                             Goat Fab'-Sp-SUC.sub.65 -RSA    ______________________________________    0        63              64    12.5     96              110    25       121             134    50       146             166    100      182             212    ______________________________________

Example 4 Indirect Sandwich Ion-capture Immunoassay for hCG

The indirect ion-capture immunoassay included the use of a solid phaseprepared substantially as described in Example 2 above, an indicatorreagent of alkaline phosphatase-sheep anti-mouse IgG conjugate (JacksonImmunoResearch Laboratories, Inc.), a capture reagent of goat anti-hCGF(ab')₂ -Sp-SUC₆₅ -RSA as prepared in Example 3, an ancillary specificbinding member of mouse monoclonal anti-hCG antibodies (ImmunoSearch;Thomas River, N.J., 08753), and a purified hCG standard solution. Theancillary specific binding member was used to bind with the analyte andthe indicator reagent. All reagents were appropriately diluted in theassay buffer. Equal volumes (150 μl) of the indicator reagent, hCGsample solution, and ancillary specific binding member were placed in aseries of test tubes. After incubation at 37° C. for five minutes, a 150μl portion of capture reagent was added to each tube. The resultingmixtures were incubated for five minutes. The assay mixture (200 μl) wasthen added to each prepared solid phase material. The solid phasematerials were then washed with washing buffer and treated with anenzyme substrate solution in the same manner as described in Example 3.above. The resulting rate of fluorescence was measured at 32.7° C. Theresults of the assay are summarized in Table 7. The results demonstratethat as the hCG test sample concentration increased there was acorresponding increase in the formation of capturereagent/analyte/ancillary specific binding member/indicator reagentcomplex, and therefore, the amount of detectable label associated withthe solid phase increased.

                  TABLE 7    ______________________________________    Ion-capture Indirect Sandwich Assay for hCG    Capture reagent: goat anti-hCG F(ab').sub.2 -Sp-SUC.sub.65 -RSA    Indicator reagent: sheep anti-mouse IgG-alkaline phosphatase    Ancillary specific binding member: mouse monoclonal anti-hCG    antibody    hCG (mlU/ml)                Rate of fluorescence (counts/sec/sec)    ______________________________________    0           13    1.5         18    3.3         27    6.3         40    12.6        70    25.0        112    50.0        230    100.0       443    200.0       732    ______________________________________

Example 5 Indirect Sandwich Ion-capture Immunoassay for hCG Using TwoAncillary Specific Binding Members

The ion-capture immunoassay protocol included the use of a solid phaseprepared substantially in accordance with the method described inExample 2, an indicator reagent of alkaline phosphatase-sheep anti-mouseIgG conjugate (Jackson lmmunoResearch Laboratories, Inc.), an ancillaryspecific binding member of mouse monoclonal anti-hCG antibodies(ImmunoSearch; Thomas River, N.J., 08753), and a purified hCG standardsolution. Additionally, the protocol used a second ancillary specificbinding member of affinity purified goat anti-hCG antibodies and acapture reagent of rabbit anti-goat IgG-Sp-SUC₆₅ -RSA. The capturereagent was prepared by coupling affinity purified rabbit anti-goat IgG(Cappel; Cochranville, Pa., 19330) to Sp-SUC₆₅ -RSA according to theprocedure described in Example 3 above. All reagents were appropriatelydiluted in the assay buffer. Equal volumes (100 μl) of the indicatorreagent, hCG sample solution, and first ancillary specific bindingmember were placed in a series of test tubes. After incubation (37° C.for ten minutes) the second ancillary specific binding member (100 μl)was added and the incubation was continued (at 37° C. for an additionalfive minutes). Finally, capture reagent (100 μl) was added to each tube.The resulting mixtures were incubated for five minutes. The assaymixture (200 μl) was then added to each prepared solid phase material.The solid phase materials were then washed with washing buffer, treatedwith enzyme substrate solution, and measured for the rate offluorescence in the same manner as described in Example 3, above. Theresults of the assay are summarized in Table 8. The results demonstratethat as the hCG test sample concentration increased there was acorresponding increase in the formation of capture reagent/ancillaryspecific binding member/analyte/ancillary specific bindingmember/indicator reagent complex, and therefore, the amount ofdetectable label associated with the solid phase increased.

                  TABLE 8    ______________________________________    Ion-capture Indirect Sandwich Assay for hCG    Capture reagent: rabbit anti-goat IgG-Sp-SUC.sub.65 -RSA    Indicator reagent: sheep anti-mouse IgG-alkaline phosphatase    Ancillary specific binding member: mouse monoclonal anti-hCG    antibody    Ancillary specific binding member: goat anti-hCG antibodies              Rate of Fluorescence (counts/sec(sec)    Goat anti-hCG (ng/ml)                hCG (40 mlU/ml)                            Negative Control (0 mlU/ml)    ______________________________________    250         3499        36    150         3708        34    50          3543        33    25          3155        30    ______________________________________

Example 6 Ion-capture Immunoassay for Anti-progesterone Antibody

a. Preparation of PGA-labeled goat anti-mouse capture reagent

The following sequence of steps describes the chemistry employed for thepreparation of an antibody/polyglutamic acid conjugate.

Conversion of PGA-sodium salt to the free acid form: The sodium salt ofPGA (200 mg; 1.47×10⁻⁵ mole; average MW 13,600; Sigma) was stirred witha cation exchange resin (AG50W-X8; 13 grams; Bio-Rad, Richmond, Calif.)in 60 milliliters of water for three hours. The supernatent wasdecanted, filtered, and evaporated providing an 80% yield of the freeacid form of PGA as a white powder (137 mg; average MW 11,620).

Preparation of isothiocyanate-PGA (ITC-PGA): To a solution of the freeacid form of PGA (65 mg; 5.6×10⁻⁶ mole) in dimethylformamide (DMF; 2 ml)was added triethylamine (100 μl; 7.2×10⁻⁴ mole) and1,4-phenylenediisothiocyanate (110 mg; 5.7×10⁻⁴ mole; Aldrich ChemicalCompany, Milwaukee, Wis.). After stirring overnight at room temperature,acetic acid (100 μl; 1.7×10⁻³ mole) was added, and the reaction mixturewas then evaporated. Methylene chloride (25 ml) was added to theresidue, and after stirring for two hours the mixture was filtered toyield the ITC-PGA as a white powder (101 mg).

The ITC-PGA (295 μg; 2.5×10⁻⁸ mole; in 40 μl of 20% DMF/0.1M sodiumphosphate at pH 7.0) was added to a buffered solution of goat anti-mouseIgG (200 μg; 1.25×10⁻⁹ mole; Sigma; in 40 μl of 0.1M sodium phosphate atpH 7) to form the PGA-labeled goat anti-mouse capture reagent. Afterstirring at room temperature for two days, 0.1M Tris (20 μl; pH 7.4) wasadded and the resulting mixture was stored at 2° to 8° C. until used.

b. Immunoassay for anti-progesterone antibody

The anti-progesterone antibody ion-capture immunoassay included the useof solid phase materials coated with a polymeric quaternary compound asdescribed in Example 1. A 60 μl sample was added to a reaction well. Thesamples consisted of a monoclonal anti-progesterone antibody atconcentrations of 0, 5, 50, 100, 250, and 500 ng/ml inphosphate-buffered saline (PBS; 50 mM sodium phosphate, 99 mM NaCl, 0.1%NaN₃, at pH 7.4). Next, 20 μl of PBS were added to the reaction well,followed by 20 μl of the buffered indicator reagent, progesteronelabeled with alkaline phosphatase (3 μg/ml in a Tris buffer of 50 mMTris, pH 7.4, 150 mM NaCl, 1% NaN₃, 1 mM MgCl₂, 0.1 mM ZnCl₂, and 1%BSA). After incubating the mixture at 34.5° C. for ten minutes, thecapture reagent was added (20 μl; PGA-labeled goat anti-mouse antibodyat a 1/100 dilution in PBS of the stock solution described above). Themixture was then incubated an additional ten minutes at 34.5° C. A 100μl aliquot of the mixture was then applied to the solid phase material,followed by three 75 μl washes of diluent. Lastly, the enzyme substratesolution (70 μl; 1.2 mM 4-methylumbelliferylphosphate in a solution of100 mM AMP, 1 mM MgCl₂, 0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3)was added to the solid phase, and the resulting rate of fluorescence wasmeasured. The results of the assay are shown in Table 9. The resultsdemonstrate that as the anti-progesterone antibody test sampleconcentration increased there was a corresponding increase in theformation of capture reagent/analyte/indicator reagent complex, andtherefore, the amount of detectable label associated with the solidphase increased.

                  TABLE 9    ______________________________________    Ion-capture Assay for Mouse Monoclonal Anti-progesterone Antibody    Capture reagent: PGA-labeled goat anti-mouse antibody    Indicator reagent: alkaline phosphatase-labeled progesterone    Anti-progesterone (ng/ml)                     Rate of fluorescence (counts/sec/sec)    ______________________________________    0                9    5                31    50               254    100              441    250              1191    500              2721    ______________________________________

Example 7 Indirect Competitive Ion-capture Immunoassay for Progesterone

The solid phase was prepared substantially in accordance with the methoddescribed in Example 1. A 60 μl sample of various concentrations ofprogesterone in PBS was mixed with 20 μl of progesterone-labeledalkaline phosphatase indicator reagent (0.4 μg/ml in the Tris buffer ofExample 4) and 20 μl of mouse anti-progesterone antibody as an ancillaryspecific binding member (0.3 μg/ml in PBS). After incubating the mixtureat 34.5° C. for ten minutes, 20 μl of of the PGA-labeled goat anti-mouseantibody capture reagent were added as described in Example 6, above.The resulting mixture was incubated an additional ten minutes at 34.5 °C. A 100 μl aliquot of the mixture was then applied to the solid phasematerial, followed by three washes of diluent. Lastly, the enzymesubstrate solution (70 μl; 1.2 mM 4-methylumbelliferylphosphate in asolution of 100 mM AMP, 1 mM MgCl₂, 0.1% NaN₃, and 4.0 mM tetramisole atpH 10.3) was added to the solid phase, and the resulting rate offluorescence was measured. The results of the assay are shown in Table10. The results demonstrate that as the progesterone test sampleconcentration increased there was a corresponding decrease in theformation of capture reagent/ancillary specific binding member/indicatorreagent complex, and therefore, the amount of detectable labelassociated with the solid phase decreased.

                  TABLE 10    ______________________________________    Ion-capture Indirect Competitive Assay for Progesterone    Capture reagent: PGA-labeled goat anti-mouse antibody    Indicator reagent: alkaline phosphatase-labeled progesterone    Ancillary specific binding member: mouse anti-progesterone antibody    Progesterone (ng/ml)                  Rate of fluorescence (counts/sec/sec)    ______________________________________    0             1203    1.88          277    3.75          145    7.5           67    15            30    30            16    ______________________________________

Example 8 Activation of Poly-L-Glutamic Acid for the Formation ofAnionic Capture Reagents

The following sequence of steps describes the chemistry used for thebulk preparation of protein-PGA conjugates for the formation ofnegatively charged capture reagents.

a. Conversion of PGA-sodium salt to the free acid form

The sodium salt of PGA (100 mg; 7.35×10⁻⁶ mole; average MW 13,600;Sigma) was stirred overnight with a hydrogen form cation exchange resin(50 equivalents/glutamate residue; AG50W-X8; Bio-Rad). The resinpreviously had been swelled and washed in distilled water, and finallyresuspended in distilled water (20 ml/7 gms dry weight of beads.) Thesupernatent was removed and lyophilized providing a 90% yield of thefree acid form of PGA (PGAFA) as a white powder (80 mg; average MW11,620). The free acid form was used to obtain solubility in organicsolvents

b. Preparation of ITC-PGAFA

The PGAFA was dissolved in solvent (DMF at ten milligrams/milliliter.) Aproton absorbing reagent (4-methyl morpholine) was added to the solutionin the amount of about one equivalent per titratable free carboxylicacid. Next, about a 100 mole excess of an amine-reactive modificationreagent (1,4-phenylene diisothiocyanate DITC! in sufficient DMF todissolve it) was added to the solution. The reaction mixture was stirredat room temperature overnight. The reaction mixture was vaporated tonear dryness, and methylene chloride (25 ml) was added to precipitatethe ITC-PGAFA. The flocculant precipitate was centrifuged, and themethylene chloride and unreacted DITC were removed. Theprecipitation/centrifugation process was repeated until substantially nodetectable DITC remained. The DITC was detected using thin layerchromatography on silica slides developed in methylene chloride;ITC-PGAFA remains at the origin, DITC moves with the solvent front. Theremaining solid was vacuum dried to yield the ITC-PGAFA as a yellowpowder.

c. Coupling of ITC-PGAFA to protein to make capture reagents

The ITC-PGAFA (at about a 1 to about a 20 mole excess to the protein)was dissolved in 0.2M sodium phosphate buffer at pH 8.5, with the volumeheld as low as possible. The pH was adjusted to 8.5 as necessary. Thedesired protein was added to this solution and incubated overnight at37° C. The preparations were then fractionated using HPLC on either ananalytical TSK 400 Bio-Rad column (7.5×300 mm, at a 1 ml/min flow rate)for 1-2 milligram protein preparations, or a TSK 4000 Beckman column(31.5×300 mm, at a 5 ml/min flow rate) for 2-10 milligram proteinpreparations. The elution buffer contained 0.1M sodium phosphate and0.3M NaCl at pH 6.8. Fractions were tested and appropriately combined.The amino acid content was determined for those fractions containingprotein so that the coupling efficiency for the various proteins atvarious coupling ratios could be determined. The results of thedeterminations are presented in Table 11.

                  TABLE 11    ______________________________________    Coupling Efficiencies of ITC-PGAFA with Various Proteins                  PGA Molar PGA Chain Percent    Protein       Excess    Number    Substitution    ______________________________________    Anti-CEA antibody                  1         0.77      77    monoclonal 1.0 mg                  5         1.7       34                  10        3.1       31                  20        8.6       43    Goat anti-rabbit antibody                  5         1.8       37    monoclonal 1.0 mg    Anti-β-hCG antibody                  10        4.6       46    monoclonal 1.0 mg                  15        5.2       36    monoclonal 10 mg                  15        7.8       52    Anti-digoxin antibody    monoclonal 1.0 mg                  15        8.1       54    monoclonal 5.0 mg                  15        5.5       37    Goat anti-mouse antibody                  15        4.3       29    polyclonal 1.0 mg    Anti-T4 antibody                  15        6.9       46    monoclonal 1.0 mg    Anti-T4 antibody                  15        13.8      92    polyclonal 7.0 mg    Rabbit Serum Albumin                  15        7.8       52    loaded with Theophylline    ______________________________________

Column 1 of Table 11 lists the quantity of protein used in the reactionsto form the various capture reagents. Column 2 lists the mole excess ofactivated ITC-PGAFA that was reacted with the Column 1 protein. Column 3provides the number of PGA chains attached per antibody by the reaction,calculated by amino acid analysis based upon a 40,000 average MW and 305repeating glutamate residues. Column 4 provides a calculation of thepercent efficiency of PGA chain substitution based upon the mole excessof activated PGA used in the reaction.

Example 9 Theophylline Ion-Capture Competitive Assay: Antigen CaptureFormat

a. Preparation of theophylline capture reagent

The activation of theophylline was accomplished by dissolvingtheophylline-butyrate (10 mg; MW 280.29; 3.57×10⁻⁵ moles) in methylenechloride (3.0 ml). A three mole excess of dicyclohexylcarbodiimide (22mg; MW 206.3) and a three mole excess of N-hydroxysuccinimide (12.3 mg;MW 115.09) were added, and the reaction mixture was stirred overnight atroom temperature. The mixture was filtered to remove dicyclohexylureaand was rotavaporated to dryness to yield ten milligrams ofN-succinimidyltheophylline-butyrate(theophylline-butyrate-oSu).

The free acid of polyglutamic acid (NH₂ -PGAFA; 1.4 mg; MW 11,798;1.19×10⁻⁷ moles) was dissolved in DMF (0.5 ml) and NMM (1.1 mg; MW101.15; 1.07×10⁻⁵ moles) The theophylline-butyrate-oSu (10 mg; at 1mg/0.5 ml DMF) was added, and the reaction mixture was stirred overnightat room temperature. Unbound theophylline was removed by dialysisagainst a 0.1M Na phosphate buffer at pH 7.0. The theophylline contentof the resulting capture reagent was analyzed, and the resultsdemonstrated that 3.9 theophylline molecules were attached per PGAchain. The theophylline-PGA capture reagent, which was capable ofbinding with anti-theophylline antibody, was then diluted to 3 μg/ml inan assay buffer containing 25 mM Tris, 100 mM NaCl, 1 mM MgCl₂, 0.1 mMZnCl₂, 0.1% NaN₃, and 1% fish gelatin at pH 7.2.

b. Preparation of the solid phase

A fiber matrix was coated with a polymeric quaternary compound toprovide the solid phase with a positive charge. Celquat® L-200 polymericcompound, a water soluble cellulose derivative, was used. A 0.5% aqueoussolution of Celquat® L-200 polymeric compound (50 μl) containing 10 mMNaCl (50 μl) was applied to the solid phase material.

c. Preparation of the indicator reagent

The indicator reagent consisted of a conjugate of alkaline phosphataseand anti-theophylline antibody, made substantially in accordance withthe protocol described in Example 3.b. The indicator reagent wasappropriately diluted (as determined by titer curve) in the assay bufferto give 0.17 micrograms of antibody/milliliter.

d. Immunoassay protocol

The indicator reagent (200 μl) was placed within a series of reactiontubes. A theophylline standard solution (200 μl; theophylline-butyratediluted to 0.6, 1.2, 2.5, 4.9, 9.9, 99.2, and 992 μg/ml in 50 mM Tris,300 mM NaCl and 0.1% NaN₃ at pH 7.2) was then added to each tube. Themixture was incubated ten minutes at 37° C. Capture reagent (200 μl) wasadded to each tube, and the reaction mixtures were incubated ten minutesat 37° C. An aliquot of each reaction mixture (200 μl) was applied tothe solid phase material, followed by one wash with diluent (75 μl). Anenzyme substrate (70 μl; 1.2 mM 4-methylumbelliferyl-phosphate in asolution of 100 mM AMP, 1.0 mM MgCl₂, 0.1% NaN₃, and 4.0 mM tetramisoleat pH 10.3) was added at 32° C. for reaction with the indicator reagent,and the resulting rate of fluorescence was measured. The results of theassay are shown in Table 12. The results demonstrate that as thetheophylline analog test sample concentration increased there was acorresponding decrease in the formation of capture reagent/indicatorreagent complex, and therefore, the amount of detectable labelassociated with the solid phase decreased.

                  TABLE 12    ______________________________________    Theophylline Ion-Capture Competitive Assay: Antigen Capture Format    Capture reagent: theophylline-PGA    Indicator reagent: alkaline phosphatase-labeled anti-theophylline    antibody    Theophylline Analog (ng/ml)                     Rate of fluorescence (counts/sec/sec)    ______________________________________    0                255    0.6              250    1.2              212    2.5              202    4.9              196    9.9              168    99.2             68    992              16    ______________________________________

Example 10 Phenylcyclidine Ion-Capture Competitive Assay-Antigen CaptureFormat

a. Preparation of Phenylcyclidine Capture Reagent

4,Hydroxy-Phenylcyclidine (1.1 mg; MW 259.37; 4.24×10⁻⁶ moles) wasdissolved in tetrahydrofuran (THF; 0.5 ml). One-half milliliter of 10%phosgene in benzene was added (130 mole excess.) The reaction wasallowed to proceed at room temperature for 2.5 hours. The solvent wasevaporated under a stream of nitrogen to yield a residue ofphenylcyclidine-4-chloroformate.

The phenylcyclidine-4-chloroformate (1.1 mg) was dissolved in THF (0.5ml). To this was added NH₂ -PGAFA (1.7 mg; MW 11,798; 1.19×10⁻⁷ moles)dissolved in 1-methyl-2-pyrrolidinone (0.5 ml). The reaction was carriedout overnight at room temperature and then rotavaporated to dryness. Theproduct was dissolved in 0.1M sodium phosphate (1.5 ml, pH 7.0). Theprecipitate was filtered, and the cloudy aqueous filtrate was extractedwith methylene chloride until clear. The phenylcyclidine-PGA capturereagent, which was capable of binding with anti-phenylcyclidineantibody, was then diluted to 5 μg/ml in an assay buffer as described inExample 9.

b. Preparation of the solid phase

The solid phase was prepared substantially in accordance with the methoddescribed in Example 9.

c. Preparation of the indicator reagent

The indicator reagent consisted of a conjugate of alkaline phosphataseand anti-phenylcyclidine antibody. The indicator reagent was diluted1/250 in the assay buffer as described in Example 9.

d. Immunoassay protocol

The indicator reagent (140 μl) was mixed with a series of samples (50 μleach) containing known amounts of phenylcyclidine (0.0, 25, 60, 120, 250and 500 ng/ml prepared in human urine), and the mixtures were incubatedfor ten minutes at 32° C. The phenylcyclidine-PGA capture reagent (100μl) was added, and the reaction mixtures were incubated for ten minutes.An aliquot of each reaction mixture (200 μl) was applied to a solidphase material. The solid phase was then washed, two times. An enzymesubstrate (70 μl; as described in Example 9) was added, and theresulting rate of fluorescence was measured. The results of the assayare shown in Table 13. The results demonstrate that as thephenylcyclidine test sample concentration increased there was acorresponding decrease in the formation of capture reagent/indicatorreagent complex, and therefore, the amount of detectable labelassociated with the solid phase decreased.

                  TABLE 13    ______________________________________    Phenylcyclidine Ion-Capture Competitive Assay: Antigen Capture    Format    Capture reagent: phenylcyclidine-PGA    Indicator reagent: alkaline phosphatase-labeled anti-phenylcyclidine    antibody    Phenylcyclidine (ng/ml)                   Rate of fluorescence (counts/sec/sec)    ______________________________________    0              570    25             133    60             60    120            33    250            18    500            9    ______________________________________

Example 11 Digoxin Ion-Capture Competitive Assay: Antigen Capture Format

a. Preparation of a digoxin-IgG-PGA capture reagent

The digoxin-IgG-PGA capture reagent was prepared substantially inaccordance with the method described in Example 8. c., with thefollowing procedural modifications. The ITC-PGA (5 mg; 1.25×10⁻⁷ mole;in 1.0 ml of 0.1M sodium phosphate at pH 8.5) was added to a bufferedsolution of rabbit IgG-digoxin (1 mg; 6.25×10⁻⁹ mole; in 1.4493 ml of0.1M sodium phosphate and 0.3M NaCl at pH 8.5) to form the capturereagent. The solution was stirred and incubated overnight at 37° C. Thepreparation was then fractionated using HPLC on a BioSil 400 (Bio-Rad300 mm×7.5 mm gel filtration column) and eluted at one milliliter/minutewith 0.1M sodium phosphate and 0.3M NaCl at pH 6.8. The digoxin-IgG-PGAcapture reagent, which was capable of binding with anti-digoxinantibody, was then diluted to 3 μg/ml in an assay buffer as described inExample 9.

b. Preparation of the solid phase

The solid phase was prepared substantially in accordance with the methoddescribed in Example 9.

c. Preparation of the indicator reagent

The indicator reagent consisted of a conjugate of alkaline phosphataseand mouse anti-digoxin antibody (ImmunoSearch; Emeryville, Calif.94608). The indicator reagent was diluted to 33.3 ng/ml in the assaybuffer as described in Example 9.

d. Immunoassay protocol

The indicator reagent (200 μl) was mixed with a series of samples (200μl) containing known amounts of digoxin (0.5, 1.0, 2.5, 5.0 and 50.0ng/ml prepared in normal human serum). The mixtures were incubated for15 minutes at 37° C. The digoxin-lgG-PGA capture reagent (200 μl) wasadded, and the reaction mixtures were incubated for 15 minutes. Analiquot of each reaction mixture (200 μl) was applied to the solid phasematerial, followed by a wash. An enzyme substrate (70 μl; as describedin Example 9) was added, and the resulting rate of fluorescence wasmeasured. The results of the assay are shown in Table 14. The resultsdemonstrate that as the digoxin test sample concentration increasedthere was a corresponding decrease in the formation of capturereagent/indicator reagent complex, and therefore, the amount ofdetectable label associated with the solid phase decreased.

                  TABLE 14    ______________________________________    Digoxin Ion-Capture Competitive Assay: Antigen Capture Format    Capture reagent: digoxin-IgG-PGA    Indicator reagent: alkaline phosphatase-labeled anti-digoxin antibody    Digoxin (ng/ml)                Rate of fluorescence (counts/sec/sec)    ______________________________________    0           115    0.5         101    1.0         91    2.5         74    5.0         60    50.0        14    ______________________________________

Example 12 Digoxin Ion-Capture Competitive Assay: Antibody CaptureFormat

a. Preparation of the indicator reagent

The indicator reagent consisted of a conjugate of alkaline phosphataseand digoxin (ImmunoSearch). The indicator reagent was diluted to 1/100in the assay buffer as described in Example 9.

b. Immunoassay protocol

The anti-digoxin-PGA capture reagent (200 μl, prepared substantially inaccordance with the protocol described in Example 8.c) was mixed with aseries of samples (200 μl each) containing known amounts of digoxin asdescribed in Example 11. The mixtures were incubated for 15 minutes at37° C. The indicator reagent (200 μl) was added, and the reactionmixtures were incubated for 15 minutes. An aliquot of each reactionmixture (200 μl) was applied to the solid phase (prepared as describedin Example 9), followed by a wash. An enzyme substrate (70 μl; asdescribed in Example 9) was added, and the resulting rate offluorescence was measured. The results of the assay are shown in Table15. The results demonstrate that as the digoxin test sampleconcentration increased there was a corresponding decrease in theformation of capture reagent/indicator reagent complex, and therefore,the amount of detectable label associated with the solid phasedecreased.

                  TABLE 15    ______________________________________    Digoxin Ion-Capture Competitive Assay: Antibody Capture Format    Capture reagent: anti-digoxin antibody-PGA    Indicator reagent: alkaline phosphatase-labeled digoxin    Digoxin (ng/ml)                Rate of fluorescence (counts/sec/sec)    ______________________________________    0           85    0.5         68    1.0         48    2.5         23    5.0         10    50.0        1    ______________________________________

Example 13 Alternative Ion-Capture Sandwich Assay for hCG

a. Preparation of the capture reagent

An anti-hCG antibody-PGA capture reagent was prepared substantially inaccordance with the method described in Example 8.c. above.

b. Preparation of the solid phase

A fiber matrix was wetted with buffer (80 μl; containing 300 mM NaCl, 50mM Tris and 0.1% NaN₃ at pH 7.5). The matrix was coated with a 0.5%aqueous solution of Celquat®L-200 polymeric compound (50 μl; containing10 mM NaCl) followed by a second wash with buffer.

c. Preparation of the indicator reagent

The indicator reagent consisted of a conjugate of alkaline phosphataseand goat anti-hCG antibody (made substantially in accordance with theprotocol described in Example 3.b). The indicator reagent wasappropriately diluted (as determined by titer curve) in assay buffercontaining 25 mM Tris, 100 mM NaCl, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.1% NaN₃,5% goat serum and 1% fish gelatin at pH 7.2.

d. Immunoassay protocol

The indicator reagent (140 μl) was mixed with a series of samples (50μl) containing known amounts of hCG in normal human serum. The mixtureswere incubated for 10 minutes at 31°-32° C. The anti-hCG antibody-PGAcapture reagent (100 μl) was added, and the reaction mixtures wereincubated for 10 minutes. An aliquot of each reaction mixture (200 μl)was applied to the solid phase material, followed by a wash. An enzymesubstrate (70 μl; as described in Example 9) was added, and theresulting rate of fluorescence was measured. The results of the assayare shown in Table 16. The results demonstrate that as the hCG testsample concentration increased there was a corresponding increase in theformation of capture reagent/analyte/indicator reagent complex, andtherefore, the amount of detectable label associated with the solidphase increased.

                  TABLE 16    ______________________________________    hCG Ion-capture Sandwich Assay    Capture reagent: anti-hCG antibody-PGA    Indicator reagent: alkaline phosphatase-labeled anti-hCG antibody                   Rate of fluorescence (counts/sec/sec)                   hCG-specific capture reagents    hCG (mIU/ml)   hCG-ITC-PGA    ______________________________________    0              22    8              38    40             116    100            236    550            644    200,000        2058    ______________________________________

Example 14 Ion-capture Flow-Through Device for a Two-Step hCG Assay

a. Preparation of the solid phase

Test sample application pads (glass fiber matrix) were treated withvarious concentrations of an aqueous solution of Merquat-100® polymericammonium compound, 100 mM Tris, 100 mM sodium chloride, 0.1% fishgelatin, 0.1% sucrose and 0.1% sodium azide. The application pads wereallowed to dry, and the pads were overlaid upon a layer of absorbentmaterial. Substantially the same procedure was used to prepare aflow-through solid phase device treated with Celquat® L-200 polymericcompound. Alternative devices were prepared by treating the applicationpad with Merquat-100® polymeric quaternary ammonium compound (a cationichomopolymer of dimethyidiallylammonium chloride, 0.5% in water)immediately before use.

b. Preparation of the indicator reagent

The indicator reagent was a conjugate of goat anti-β-hCG antibody andalkaline phosphatase, diluted in 1% Brij®⁻ 35 polyoxyethylene (23)lauryl ether (Sigma), 100 mM Tris, 500 mM NaCl, 1 mM MgCl₂, 0.1 mMZnCl₂, 0.1% NaN₃ and 0.5% non-fat dry milk at pH 7.2. The indicatorreagent was filtered through a 0.22 μm filter before use.

In alternative indicator reagent preparations, dextran sulfate (MW5,000) or heparin was included as a nonspecific binding blocker. Theblocker was used to enhance the signal-to-noise ratio by inhibiting thebinding of the labeled antibody to non-analyte.

c. Preparation of the capture reagent

A monoclonal anti-β-hCG antibody-PGA capture reagent was preparedsubstantially in accordance with the method described in Example 8.c.above. Every five milliliters of the coupling reaction mixture wasfractionated on a gel filtration chromatography column (2.4×54 cm, at a0.4 ml/minute flow rate). The elution buffer contained 0.1M sodiumphosphate, 0.3M NaCl and 0.05% NaN₃, at pH 8.5. The polymericanion/antibody conjugate was diluted with 25 mM Tris, 100 mM NaCl, 1 mMMgCl₂, 0.1 mM ZnCl₂, 0.1% NaN₃, 10% normal mouse serum and 1% fishgelatin at pH 7.2. The capture reagent was filtered through a 0.22 μmfilter before use.

d. Immunoassay protocol

The capture reagent (80 μl) was mixed with an equal volume of testsample containing a known amount of hCG in normal human serum. Themixture was incubated at approximately 31°-32° C. for approximatelytwelve minutes. The specific binding reaction resulted in the formationof a capture reagent/analyte complex.

Each reaction mixture (80 μl) was then applied to a flow-through device,followed by a wash with Tris buffered saline (75 μl). The indicatorreagent (50 μl) was then applied to the solid phase device and incubatedfor twelve minutes. The device was then washed two times.

An enzyme substrate (70 μl; 1.2 mM 4-methylumbelliferyl-phosphate in asolution of 100 mM AMP, 0.01% EDTA, 0.1% NaN₃, and 4.0 mM tetramisole atpH 10.3) was added, and the resulting rate of fluorescence was measured.The results of the assay are shown in Tables 17-19. The resultsdemonstrated that as the hCG test sample concentration increased therewas a corresponding increase in the formation of capturereagent/analyte/indicator reagent complex, and therefore, the amount ofdetectable label associated with the solid phase increased. The resultsshow that the signal to noise ratio is improved by including anonspecific binding blocker in the indicator reagent. Furthermore, theresults demonstrated that the cationic homopolymer ofdimethyldiallylammonium chloride was a preferred polymeric cation forthe preparation of the solid phase for use in two-step assays whereinthe device is subjected to one or more washings, e.g., the Merquat-100®polymeric ammonium compound has a nitrogen content of about 10%(exclusive of counter ion), whereas the Celquat® H-100 polymericcompound has a nitrogen content of about 1% (exclusive of counter ion).

                  TABLE 17    ______________________________________    hCG Ion-capture two-step Sandwich Assay    Capture reagent: anti-β-hCG antibody-PGA (0.5 μg/test)    Indicator reagent: alkaline phosphatase-labeled anti-β-hCG antibody    (with and without nonspecific binding blocker)    Solid phase: coated with a cationic homopolymer of    dimethyldiallylammonium chloride immediately before use               Rate of fluorescence (counts/sec/sec)    hCG (mIU/ml) 2% dextran sulfate                              no blocker    ______________________________________    0            68           255    100          1028         1104    ______________________________________

                  TABLE 18    ______________________________________    hCG Ion-capture two-step Sandwich Assay    Capture reagent: anti-β-hCG antibody-PGA (0.5 μg/test)    Indicator reagent: alkaline phosphatase-labeled anti-β-hCG antibody    (with blocker)    Solid phase: with varying cationic polymer concentration           Rate of fluorescence (counts/sec/sec)           Merquat-100 ® polymeric ammonium compound           (% w/v)    hCG (mIU/ml)             0.02     0.04     0.2    0.4    0.6    ______________________________________    0        34       31       26     30     39    100      514      578      627    661    647    ______________________________________

                  TABLE 19    ______________________________________    hCG Ion-capture two-step Sandwich Assay    Capture reagent: anti-β-hCG antibody-PGA    Indicator reagent: alkaline phosphatase-labeled anti-β-hCG antibody    Solid phase: with 0.125% Celquat ® H-100 polymeric compound             Rate of fluorescence (counts/sec/sec)             quantity of capture antibody (μg/test)    hCG (mIU/ml)               0.268       0.402     0.652    ______________________________________    0          86          100       115    100        186         202       259    ______________________________________

Example 15 Ion-capture Flow-Through Device for Thyroid StimulatingHormone (TSH) Assay

a. Preparation of the solid phase

An application pad (glass fiber matrix) was treated with an aqueoussolution of Merquat-100® polymeric ammonium compound substantially inaccordance with the procedure described in Example 14.a. The pad wasthen overlaid upon a layer of absorbent material to complete theflow-through solid phase device.

b. Preparation of the indicator reagent

The indicator reagent was a conjugate of goat anti-β-hCG antibody andalkaline phosphatase, diluted in 1% Brij®⁻ 35 polyoxyethylene (23)lauryl ether, 1% fish gelatin, 100 mM Tris, 500 mM NaCl, 1 mM MgCl₂, 0.1mM ZnCl₂, 0.1% NaN₃ and 0.5% non-fat dry milk at pH 7.2. The indicatorreagent was filtered through a 0.22 μm filter before use. Dextransulfate (0.5%, MW 5,000) was added as a nonspecific binding blocker.

c. Preparation of the capture reagent

The capture reagent was prepared by coupling a Protein A purifiedmonoclonal anti-TSH antibody with carboxymethyl amylose (CMA;Polysciences, Inc., Warrington, Pa.).

Coupling was performed using a water-soluble carbodiimide reagent(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; EDCI) substantially inaccordance with the following procedure.

The coupling mixture contained an antibody solution (2 ml; 1 mg/ml inMES buffer 25 mM, 2-(N-Morpholino)ethanesulfonic acid! pH 5.5) and CMA(1.6 ml; 10 mg/ml in MES buffer). To the solution was added, withstirring, a freshly prepared EDCI solution (40 μl; 100 mg/ml in MESbuffer). The reaction mixture was stirred at room temperature for 40minutes. The reaction was quenched by adding a 25% glycine solution (67μl), and the product was then fractionated by gel filtrationchromatography using a TSKgel G4000SW column (2.15 cm×30 cm) fitted witha TSKguard column SW (2.15 cm×7.5 cm; Anspec Co., Ann Arbor, Mich.). Thecolumn was eluted with PBS (0.1M sodium phosphate, 0.3M NaCl and 0.05%sodium azide, at pH 6.8). The purified Antibody/CMA capture reagent wasdiluted in a diluent containing 50 mM Tris, 300 mM NaCl, 1% bovine serumalbumin, 2.5% fish gelatin and 0.1% NaN₃, at pH 7.5.

d. Immunoassay protocol

The capture reagent (30 μl) and Tris buffered saline (100 μl; 500 mMTris, 300 mM NaCl and 0.1% NaH₃) were mixed with a test sample (50 μl)containing a known amount of hCG in normal human serum. The reactionmixture was incubated at approximately 33°-34° C. for approximately tenminutes. The specific binding reaction resulted in the formation of acapture reagent/analyte complex.

An aliquot of each reaction mixture (140 μl) was applied to a solidphase device, followed by a wash with Tris buffered saline (150 μl). Theindicator reagent (70 μl) was applied to the device and incubated forapproximately ten minutes. The device was then washed two times withbuffer (100 μl each). The enzyme substrate (70 μl; 1.2 mM4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 0.01% EDTA,0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3) was added, and theresulting rate of fluorescence was measured.

The results of the assay are shown in Table 20. The results demonstratedthat as the concentration of TSH in the test sample increased, there wasa corresponding increase in the formation of capturereagenVanalyte/indicator reagent complex. Therefore, the amount ofdetectable label associated with the solid phase increased as theconcentration of analyte increased. The results also demonstrated thatthe combination of Merquat-100® polymeric ammonium compound withpolyacrylic acid or with carboxymethylamylose provided a solid phase andcapture reagents which were advantageously used in two-step assayswherein the device is subjected to one or more washings ormanipulations.

                  TABLE 20    ______________________________________    TSH Ion-capture Two-step Sandwich Assay    (using polyacrylic acid or carboxymethylamylose polyanions)              Rate of fluorescence (counts/sec/sec)    TSH (mIU/ml)                carboxymethylamylose                              polyacrylic acid    ______________________________________    0           7.1           6.4    0.5         13.3          12.1    2.0         34.7          28.7    10.0        147.5         119.8    40.0        513.9         442.6    100.0       1121.6        995.5    ______________________________________

e. TSH capturing efficiency

Radioiodinated TSH was used in the assay protocol, as described inExample 15.d, to demonstrate the more efficient TSH capturing ofCMA-coupled antibodies than that of polyaspartic- andpolyglutamic-coupled antibodies. The coupling of antibodies to thepolyanions was performed substantially in accordance with the methoddescribed above (Example 15.c.) After the rate of fluorescence wasmeasured at the end of the assay protocol, the radioactivity of TSHcaptured on the solid phase material was also measured by means of ascintillation spectrometer (Auto-Logic, Abbott Laboratories, NorthChicago, Ill.). The results of this procedure are demonstrated in Table20 (a).

                  TABLE 20 (a)    ______________________________________    Capture of Radiolabeled TSH in the Cationic Solid Phase Material    Polyanion-coupled anti-TSH  Rate of fluorescence    antibody       % TSH captured                                (counts/sec/sec)    ______________________________________    Carboxymethylamylose                   70           662    Polyaspartic Acid                   1.5          37    Polyglutamic Acid                   2.0          57    ______________________________________

Example 16 Ion-capture Flow-Through Device for a One-Step hCG Assay

a. Preparation of the solid phase

A glass fiber matrix was treated with an aqueous solution ofMerquat-100® polymeric ammonium compound substantially in accordancewith the procedure described in Example 14. a, above. The pad was thenoverlaid upon a layer of absorbent material to complete the device.

b. Preparation of the indicator reagent

The indicator reagent was a goat anti-β-hCG antibody conjugated toalkaline phosphatase and diluted in 3.33% Brij®⁻ 35 polyoxyethylene (23)lauryl ether, 5 mM Tris, 1 mM MgCl₂, 0.1 mM ZnCl₂, 0.1% NaN₃ and 5% fishgelatin at pH 7.2. The indicator reagent was filtered through a 0.2 μmfilter before use. In alternative indicator reagent preparations,carboxymethyl cellulose (MW 250,000) or carboxymethyl dextran wasincluded as a nonspecific binding blocker.

c. Preparation of the capture reagent

A monoclonal anti-hCG antibody-PGA capture reagent was preparedsubstantially in accordance with the method described in Example 15.c,above. The polymeric anion/antibody conjugate was diluted with 3.33%Brij®⁻ 35 polyoxyethylene (23) lauryl ether, 5 mM Tris, 500 mM NaCl, 1mM MgCl₂, 0.1 mM ZnCl₂, 0.1% NaN₃, and 5% fish gelatin at pH 7.2. Theenzyme substrate was 1.2 mM 4-methylumbelliferyl-phosphate in a solutionof 100 mM AMP, 0.01% EDTA, 0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3.

d. Immunoassay protocol

The capture reagent (50 μl), indicator reagent (55 μl) and samplediluent buffer (35 μl: 75% normal calf serum, 25% normal goat serum and0.2% NaN₃, filtered through a 0.22 μm filter before use) were mixed witha test sample (30 μl) containing a known amount of hCG in normal humanserum. The mixture was incubated at approximately 33°-34° C. forapproximately fourteen minutes. The specific binding reaction resultedin the formation of a capture reagenVanalyte/indicator reagent complex.

An aliquot of each reaction mixture (110 μl) was then applied to a solidphase device, followed by two washes with Tris buffered saline (75 μl).The enzyme substrate (65 μl) was added, and the resulting rate offluorescence was measured.

The results of the assay are shown in Table 20. The results demonstratedthat as the hCG test sample concentration increased there was acorresponding increase in the formation of capturereagent/analyte/indicator reagent complex, and therefore, the amount ofdetectable label associated with the solid phase increased. Furthermore,the results show that the signal to noise ratio was improved when a freepolyanionic substance was included in the indicator reagent as anonspecific binding blocker, even though the capture reagent was apolymeric anion/antibody conjugate.

                  TABLE 21    ______________________________________    hCG Ion-capture Sandwich Assay    Capture reagent: anti-hCG antibody-PGA    Indicator reagent: alkaline phosphatase-labeled anti-hCG antibody    hCG (mIU/ml)                0        0.01      0.25  0.5    ______________________________________              Rate of fluorescence (counts/sec/sec)              % carboxymethyl cellulose in indicator              reagent    0           37.2     23.8      17.2  13.3    10          76.8     58.4      48.8  42.1    1000        1803.6   1665.4    1692.2                                         1507.2              Rate of fluorescence (counts/sec/sec)              % carboxymethyl dextran in indicator              reagent    0           35.6     30.0      17.8  14.8    10          75.2     68.4      54.7  49.8    1000        1826.6   1851.2    1739.5                                         1646.6    ______________________________________

Example 17 Ion-Capture Teststrip for an hCG Sandwich Assay

a. Preparation of the solid phase

A rectangular zone on a central portion of a strip of nitrocellulose (5μm pore size;

Schleicher & Schuell; Dassel, Germany) was treated with an aqueoussolution of 0.05% Merquat-100® polymeric ammonium compound and 10 mMTris to form a positively charged capture or detection zone.

b. Preparation of the indicator reagent

The indicator reagent was made of colloidal selenium particles coatedwith mouse monoclonal anti-hCG antibody. The indicator reagent wasappropriately diluted (as determined by titer curve) in assay buffercontaining 50 mM Tris, 2% lactose, 2% casein, 1% goat serum and 1% mouseserum at pH 8.4.

c. Preparation of the capture reagent

A goat anti-β-hCG antibody was coupled to poly-L-glutamic acid using1-ethyl-3-(3-dimethylaminopropyl)-carbodimide substantially inaccordance with the method described in Example 15.c, above. The capturereagent was then appropriately diluted in the same diluent as theindicator reagent.

d. Immunoassay protocol

The indicator reagent (50 μl) was mixed with an equal volume of capturereagent. The mixture was then combined with a series of samples (0, 50,100 and 250 mlU/ml; 150 μl each) containing known amounts of hCG innormal human urine. The resultant reaction mixtures were incubated forfive minutes at room temperature. The specific binding reaction resultedin the formation of a capture reagent/analyte/indicator reagent complex.

Each reaction mixture (250 μl) was then applied to one end of theprepared strip of nitrocellulose. The mixture was allowed to migratethrough the strip to the capture zone and through the zone. Capturereagent and complexes thereof were retained at the capture zone, whereinthe indicator reagent complexed with the retained capture reagentindicated the amount of analyte in the test sample as well as the thepresence of analyte in the test sample. The 0 mlU/ml test sampleproduced no coloration of the capture zone. The 50, 100 and 250 mlU/mltest samples produced visible coloration of the capture zone.

Example 18 Ion-Capture Teststrip Device for an hCG Assay

a. Preparation of the solid phase

A rectangular zone on a central portion of a strip of nitrocellulose (5μm pore size;

Schleicher & Schuell) was treated with an aqueous solution of 1%Celquat® L-200 polymeric compound to form a positively charged capturezone. The cationic polymer was dispensed using a #29 gauge tube (MICROInc., Elmhurst, N.Y.) moving at a rate of 0.5 inches/second with a flowrate of 0.05 milliliter/minute.

b. Preparation of the indicator reagent

The indicator reagent was made of colloidal selenium particles coatedwith mouse monoclonal anti-hCG antibody. The indicator reagent wasappropriately diluted (as determined by titer curve) in assay buffercontaining 50 mM Tris, 2% lactose, 2% casein, 1% goat serum and 1% mouseserum at pH 8.4.

c. Preparation of the capture reagent

An anti-β-hCG antibody was coupled with poly-glutamic acid substantiallyin accordance with the method described in Example 8.c, above.

d. Preparation of the assay device

A reagent pad or test sample application pad was prepared by soaking apad of absorbant material (40 μpore glass fiber material; Lydall Inc.,Hamptonville, N.C.) with a mixture containing the capture reagent (20μg/ml) and the indicator reagent (antibody concentration 0.024 mg/mL,selenium concentration 0.3 mg/mL) in Tris buffered saline (0.1M Tris,0.9% NaCl, pH 7.8), 1.0% casein. The pad was then air dried. Theteststrip device was then constructed by contacting the test sampleapplication pad and nitrocellulose strip, and then double laminating thepad and nitrocellulose so that the application pad overlapped at leastan end portion of the nitrocellulose strip offset from the capture zone.

e. Immunoassay protocol

A test sample containing a known amount of hCG in normal human urine (0,50 and 250 mlU/ml; 50 μl each) was applied to the test sampleapplication pad of the assay device, or the application pad was dippedinto the test sample. The test sample, resolubilized assay reagents andcomplexes thereof migrated from the application pad to and through thenitrocellulose strip. After five minutes, at room temperature, thespecific binding reaction and the ion-capture reaction resulted in theformation of a capture reagent/analyte/indicator reagent complex whichwas immobilized at the capture zone of the teststrip. Unbound indicatorreagent and test sample components passed through the capture zone. The0 mlU/ml test sample produced no detectable signal at the capture zone.The 50 mlU/ml test sample produced a faintly detectable visible signalat the capture zone. The 250 mlU/ml test sample produced a stronglydetectable visible signal at the capture zone. The assay results alsodemonstrated that a homogeneous specific binding reaction could form atertiary complex while reacting in a solid phase teststrip device.

Example 19 Ion-Capture Teststrip Device for a Phenylcyclidine (PCP)Assay

a. Preparation of the solid phase

A rectangular zone on a central portion of an elongated strip ofnitrocellulose (3 mm in width) was treated with an aqueous solution of0.5% Merquat-100® polymeric ammonium compound to form a positivelycharged capture zone.

b. Preparation of the indicator reagent

The indicator reagent was made of colloidal selenium particles coatedwith PCP antibody.

c. Preparation of the capture reagent

A PCP antigen was conjugated to poly-glutamic acid substantially inaccordance with the method described in Example 10.a, above.

d. Preparation of the assay device

An assay reagent pad (3 mm in width) or test sample application pad wasprepared by soaking an absorbant material (Whatman PD075 glass fiberfilter; Whatman Specialty Papers, Clifton, N.J.) with the indicatorreagent (2.5 mg/ml; 4% casein, 4% sucrose, 1% polyethylene glycol MW15,000-25,000! in 0.01M Tris). The application pad was then air dried.The application pad and nitrocellulose where then assembled so that thereagent pad overlapped one end of the nitrocellulose by approximatelyone millimeter.

e. Immunoassay protocol

The capture reagent (15 μl) and an equal volume of test sample,containing a known dilution of PCP in distilled water (1:10, 1:100,1:1000, 1:10000), were mixed. The mixture was applied to the test sampleapplication pad. The mixture was allowed to migrate through the pad andstrip for at least ten five minutes. The competitive binding reactionresulted in the formation of capture reagent/indicator reagent complexand indicator reagent/analyte complex, wherein the amount of capturereagent/indicator reagent complex decreased as the amount of analyte inthe test sample increased. The polyelectrolyte reaction resulted in theimmobilization of the capture reagent/indicator reagent complex in thecapture zone of the teststrip. Unbound indicator reagent and unreactedtest sample components, as well as indicator reagent/analyte complex,passed through the capture zone. The assay results demonstrated that thehigher the amount of PCP in the test sample, the lower the detectablesignal at the capture zone. The assay results also demonstrated that ahomogeneous specific binding reaction could take place in a solid phaseteststrip device.

Example 20 Ion-Capture Flow-through Device for an hCG Assay

a. Preparation of the solid phase

A glass fiber filter material was treated with an aqueous solution of0.125% Celquat® L-200 polymeric quaternary ammonium compound to form apositively charged capture zone. The glass fiber filter was then setupon a second layer of absorbent material which serves to pick up excessreagents and test sample which pass through the layer containing thecharged detection zone.

b. Preparation of the indicator reagent

The indicator reagent was made of colloidal gold particles coated withaffinity purified goat anti-β-hCG antibody. A solution containing goldchloride (100 mg) in distilled water (510 ml) was heated to boiling andmixed with 1% sodium citrate (8.0 ml). The heat was removed when thecolor of the solution changed from yellow to dark red (approximatelythree minutes). The solution was cooled to room temperature by flushingunder tap water. A portion (10 ml) of the resultant gold colloid wastitrated with 150 millimolar borate buffer (pH 9.0) to pH 7.0.

Fifty microliters of goat anti-β-hCG antibody (9 mg/ml) was added to thegold colloid and mixed at room temperature for one minute. The mixturewas then treated with 10% bovine serum albumin (300 μl) and centrifugedat 14,000 rpm for one minute. The bottom layer of the colloid/antibodymixture (approximately 320 μl) was recovered for use as the indicatorreagent.

c. Preparation of the capture reagent

Purified monoclonal anti-hCG antibodies were modified with ITC-PGAsubstantially in accordance with the methods described in Examples 6 and8, above.

d. Immunoassay protocol

All reagents were appropriately diluted in an assay buffer containing 50mM Tris, 150 mM NaCl, pH 7.5 and 3% casein. A test sample (50 μl)containing a known amount of hCG in normal human urine (0, 25, 50, 100and 250 mlU/ml) was mixed with an equal volume of indicator reagent, andthe mixture was incubated at room temperature for five minutes. Capturereagent (50 μl) was then added to the mixture. The resulting mixture wasthen transferred to the solid phase that had been pre-wetted with buffer(80 μl). The flow-through devices were then rinsed twice with buffer. Avisible purple color was detected for those devices which receivedhCG-containing reaction mixtures, while the 0 mlU/ml test sampleproduced no detectable signal at the capture zone. The darkness of thesignal at the capture zone increased with the increase of hCGconcentration.

Example 21 Competitive Digoxin Assay Using Ion-Capture

a. Preparation of the solid phase

Test sample application pads (glass fiber matrix) were overcoated withvarious concentrations of an aqueous solution of Merquat-100® polymericammonium compound, 100 mM Tris, 100 mM sodium chloride, 0.1% fishgelatin, 0.1% sucrose and 0.1% sodium azide. The application pads wereallowed to dry and were then overlaid upon a layer of absorbent materialto prepare flow-through devices.

b. Preparation of the indicator reagent

The indicator reagent was a conjugate of digoxin dialdehyde and alkalinephosphatase, diluted in 50 mM Tris, 100 mM NaCl, 1 mM MgCl₂, 0.1 mMZnCl₂, 0.1% NaN₃ and 0.1% bovine serum albumin at pH 7.5.

c. Preparation of the capture reagent

The first capture reagent, a goat anti-digoxin antibody coupled to1,4-phenylene diisothiocyanate activated poly-L-aspartic acid (ITC-PAA),was prepared substantially in accordance with the method described inExample 8.c. above. Poly-L-aspartic acid was used in place ofpoly-L-glutamic acid.

A second capture reagent was made of rabbit anti-goat IgG antibodycoupled to poly-L-aspartic acid using EDCI substantially in accordancewith the coupling protocol described in Example 15.c, above. Ananalyte-specific ancillary binding member (goat anti-digoxin antibody)was used together with this capture reagent to bind the analyte to thesolid phase. In one embodiment, the capture reagent was a preformedcomplex of the negatively chaanti-ganti-goat antibody and the goatanti-digoxi reagents were appropriately diluted before use with 50 mMTris, 50 mM NaCl, 0.1% NaN₃ and 0.1% bovine serum albumin at pH 7.5

d. Immunoassay protocol

In assays using the first capture reagent, or direct capture system, thecapture reagent (60 μl) was mixed with test sample (18 μl) containing aknown amount of digoxin. The reaction mixture was incubated atapproximately 33°-34° C. for about ten minutes. The specific bindingreaction resulted in the formation of a capture reagent/analyte complex.The indicator reagent (60 μl) was then added to the reaction mixture,and the mixture was incubated for about another eleven minutes. Thespecific binding reaction resulted in the formation of capturereagent/indicator reagent complex in proportion to the amount of analytepresent in the test sample. A portion of each reaction mixture (80 μl)was then applied to the solid phase, followed by two washes with Trisbuffered saline (75 μl). An enzyme substrate (70 μl; 1.2 mM4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 0.01% EDTA,0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3) was added, and theresulting rate of fluorescence was measured.

In an indirect assay using the second capture reagent, the preformedcapture reagent/ancillary binding member complex (50 μl), indicatorreagent (55 μl) and digoxin test sample (25 μl) were combined withsample diluent buffer (91 μl). The mixture was incubated forapproximately nine minutes. The specific binding reaction resulted inthe formation of capture reagent/ancillary binding member/analytecomplex and capture reagent/ancillary binding member/indicator reagentcomplex in proportion to the amount of analyte present in the testsample. An aliquot of the reaction mixture (180 μl) was then applied tothe solid phase, followed by two washes with Tris buffered saline (75μl). The enzyme substrate (70 μl) was added, and the resulting rate offluorescence was measured.

The results of the assay are shown in Table 22. The results demonstratedthat as the digoxin test sample concentration increased there was acorresponding decrease in the formation of complex containing indicatorreagent. Therefore, the amount of detectable label associated with thesolid phase decreased with the increase of digoxin in the test sample.

                  TABLE 22    ______________________________________    Digoxin Ion-capture Competitive Assay    ______________________________________                Semi-sequential    Protocol:   One-Step      One-Step    Precoated Solid Phase:                0.2% Merquat-100 ®                              0.2% Merquat-100 ®    Concentration of                162 ng Goat   90 ng Rabbit anti-Goat/    Antibody/Test:                anti-Digoxin  64 ng Goat anti-Digoxin    Indicator Reagent:                Alkaline Phosphatase/                              Alkaline Phosphatase/                Digoxin Conjugate                              Digoxin Conjugate    ______________________________________                Direct        Indirect    Digoxin (ng/ml)                Rate of fluorescence (counts/sec/sec)    ______________________________________    0           680           456    0.5         546           387    1           413           309    2           303           247    3           261           179    5           183           121    ______________________________________

Example 22 Ion-capture Flow-Through Device for a Total T3(Triiodothyronine) Competitive Assay

a. Preperation of the solid phase

Test sample application pads (glass fiber matrix) were treated withvarious concentrations of an aqueous solution of Celquat® L-200polymeric quaternary ammonium compound or Merquat-100® polymericammonium compound, 100 mM Tris, 100 mM sodium chloride, 0.1% fishgelatin, 0.1% sucrose and 0.1% sodium azide. The application pads wereallowed to dry, and the pads were overlaid upon a layer of absorbentmaterial to form the individual assay devices.

b. Preparation of the indicator reagent

The indicator reagent was a conjugate of T3 and alkaline phosphatase,diluted in 50 mM Tris, 100 mM NaCl, 1.0 mM MgCl₂, 0.1 mM ZnCl₂ and 1.0%bovine serum albumin at pH 7.5. Dextran sulfate (MW 5,000) was includedas a nonspecific binding blocker. The blocker was used to enhance thesignal-to-noise ratio by inhibiting the binding of the labeled antibodyto non-analyte.

c. Preparation of the capture reagent

The capture reagent, an anti-T3 antibody coupled to polyaspartic acid(PAA-anti-T3 antibody), polyacrylic acid (PAcA-anti-T3 antibody) orcarboxymethyl cellulose (CMA-anti-T3 antibody) anionic polymermolecules, was prepared substantially in accordance with the methoddescribed in the Example 15.c EDCI coupling method, with the exceptionthat no chromatographic filtration of the capture reagent was performed.The capture reagent was diluted with 800 mM Tris, 50 mM NaCl, 0.1% NaN₃,0.01% furosemide, 0.1% Tween-20,1.0% bovine serum albumin and 0.08 mg/mlgoat IgG at pH 7.4.

d. Immunoassay protocol

The capture reagent (50 μl) was mixed with an equal volume of testsample, containing a known amount of Total T3, and sample diluent buffer(150 μl). The reaction mixture was incubated for approximately 15minutes. The specific binding reaction resulted in the formation of acapture reagent/analyte complex.

Each reaction mixture (150 μl) was then applied to a solid phase. Theindicator reagent (60 μl) was then applied to the solid phase andincubated for eight minutes. The device was then washed two times. Anenzyme substrate (50 μl) was added, and the resulting rate offluorescence was measured.

In an alternative assay format, the solid phase was also washed prior tothe addition of the indicator reagent. In yet another assay format, thecapture reagent and test sample were combined and incubated, followed bythe addition of indicator reagent and further incubation prior toplacing an aliquot of the reaction mixture on the solid phase.

The polyelectrolyte interaction of the capture reagent and theoppositely charged solid phase resulted in the immobilization of capturereagent and capture reagent complexes on the solid phase devices. Anenzyme substrate (70 μl; 1.2 mM 4-methylumbelliferyl-phosphate in asolution of 100 mM AMP, 0.01% EDTA, 0.1% NaN₃, and 4.0 mM tetramisole atpH 10.3) was added, and the resulting rate of fluorescence was measured.

In each assay, the results demonstrated that as the Total T3 test sampleconcentration increased there was a corresponding increase in theformation of capture reagent/analyte complex, and therefore, the amountof detectable label associated with the solid phase decreased.Furthermore, the results show that the signal to noise ratio is improvedby including a nonspecific binding blocker, dextran sulfate, in theindicator reagent.

                  TABLE 23    ______________________________________    Total T3 Competitive Assay    Calibration data comparing one-step and two-step assay protocols    ______________________________________    Protocol:    One-step     Two-step    Precoated Solid Phase:                 0.5% Celquat ®                              0.2% Merquat-100 ®    Capture Antibody:                 ITC-PGA anti-T3                              EDAC-PAA anti-T3    (per test)   antibody (0.25 μg)                              antibody (0.02 μg)    Indicator Reagent:                 no blocker   with 0.1% dextran sulfate    ______________________________________    Calibrators concentration    ng/ml total T3                 Rate of fluorescence (counts/sec/sec)    ______________________________________    0            518          616    0.5          386          513    1.0          310          403    2.0          218          260    4.0          123          109    8.0          71           48    ______________________________________

                  TABLE 24    ______________________________________    Total T3 Competitive Two-Step Assay    comparison of indicator reagents with and without a nonspecific binding    blocker    ______________________________________    Precoated Solid Phase:                 0.2% Merquat-100 ®                               0.2% Merquat-100 ®    Capture Antibody:                 EDAC-PAA anti-T3                               EDAC-PAA anti-T3    (per test)   antibody (0.02 μg)                               antibody (0.02 μg)    Indicator Reagent:                 no blocker    with 0.1% dextran    T3 alkaline phosphatase    sulfate    dilution:    1:400         1:150    ______________________________________    Calibrators concentration    ng/ml total T3                 Rate of fluorescence (counts/sec/sec)    ______________________________________    0            641           536    2.0          361           220    8.0          81            39    ______________________________________

                  TABLE 25    ______________________________________    Total T3 Competitive Assay    Calibration data comparing different T3 capture reagents    ______________________________________    Capture Antibody:               PAA-anti-T3                          PAcA-anti-T3                                     CMA-anti-T3    (per test) antibody (0.013                          antibody (0.015                                     antibody (0.013               μg)     μg)     μg)    ______________________________________    Calibrators    concentration    ng/ml total T3               Rate of fluorescence (counts/sec/sec)    ______________________________________    0          509        544        507    0.5        401        443        394    1.0        332        344        322    2.0        203        219        204    4.0        94         107        99    8.0        47         57         51    ______________________________________

Example 23 HIV-1 Anti-p24 antibody Detection Using an Ion-CaptureSandwich Assay

The solid phase devices were prepared by overcoating glass fibermatrixes with a polycationic substance and overlaying the matrixes uponan absorbent material. The capture reagent, was prepared by the covalentcoupling of a polyanionic substance to purified recombinant p24 antigen.The indicator reagent was a conjugate of alkaline phosphatase andanti-biotin antibody which bound to the analyte antibody by means of ananalyte-specific ancillary specific binding member, i.e., biotinylatedp24 antigen. The enzyme substrate was 4-methylumbelliferyl-phosphate.

The capture reagent was reacted with the test sample to form a capturereagent/analyte complex. Excess reagent and test sample components wereremoved and the complex was immobilized by passage through theoppositely charged solid phase. The amount of captured analyte was thendetermined by the sequential addition of the ancillary specific bindingmember, indicator reagent and enzyme substrate.

Example 24 Ion-Capture Device with Procedural Control

In an alternative embodiment, the solid phase reaction matrix of Example17 was prepared such that two assay reagents were incorporated into thematrix in an overlapping design to form the detection zone. The reactionof one reagent completed one portion of a detectable pattern, and thereaction of a second reagent completed another portion of the detectablepattern.

For example, the anionic polymer (such as polyglutamic acid) was appliedto the solid phase to form the vertical bar of a "cross" shaped design.The anionic polymer attracted and attached to the oppositely chargedcapture reagent comprising an analyte-specific binding member conjugatedto a polymeric cation. The reaction of the capture reagent, analyte andan indicator reagent specific for the analyte resulted in a detectablecomplex being immobilized at the vertical bar.

A procedural control reaction zone, which did not involve an analytereaction, was formed in the shape of the horizontal bar of thecross-shaped detection zone. A reagent which reacted with andimmobilized the indicator reagent without the formation of ananalyte-containing complex was used.

For example, when the indicator reagent was made of colloidal goldparticles coated with affinity purified goat anti-β-hCG antibody, thenthe horizontal bar of the cross-shaped detection zone included aspecific binding member which would directly bind to the goat anti-β-hCGantibody, e.g., a rabbit anti-goat antibody. Thus, detectable label wasimmobilized in the horizontal bar whether or not there was analytepresent in the test sample.

Example 25 Rubella antibody Detection Using an Ion-Capture SandwichAssay

The solid phase devices were prepared by coating glass fiber matrixeswith a polycationic substance, such as 0.125% (w/v) aqueous solution ofa polymeric quaternary ammonium compound, and overlaying the matrixesupon an absorbent material. The capture reagent, was prepared theGilchrist strain of Rubella. The virus was propagated in BHK-21 cellsfor three days in roller bottles. The medium was harvested and cellulardebris was removed by continuous flow centrifugation. Clarified mediumwas concentrated approximately 12 times using a hollow fiber membrane(Amicon DC30 and PM10; Amicon Corporation, Scientific Systems Division,Danvers, Mass.). The virus was pelleted from the concentrate byultracentrifugation with the time and speed adjusted to clear the mediumof all material 80s (sedimentation coefficient) and larger. Viruspelleted from 100 milliliters of concentrate was added to twomilliliters of PBS/EDTA buffer and solubilized by sonication. Theresultant preparations typically had a protein concentration of from twoto three milligrams/milliliter and a hemagglutination titer ofapproximately 20,000.

Calibrators containing 0, 15, 50, 100, 200 and 500 International Units(IU) IgG antibody/milliliter were prepared by appropriately dilutingpositive human sera in negative human sera. The calibrators werereferenced to the World Health Organization International Standard forAnti-Rubella Serum Adjusted to 500 IU/milliliter.

The assay was performed at 31°-32° C. An aliquot (20 μl) of five-folddiluted calibrators containing 0, 15, 50, 100, 200 and 500 IU/mi ofhuman IgG anti-Rubella was incubated with Rubella virus (133 μl) fornine minutes. After the addition of buffer (85 ml), the diluted testsolution (160 μl) was contacted to the solid phase. The solid phase wasthen washed with buffer, followed by the addition of an indicatorreagent (50 μl; a conjugate of alkaline phosphatase and goat anti-humanIgG). Five minutes after the indicator reagent was added, the solidphase was rinsed with buffer (250 μl total). An enzyme substrate (70 μl;1.2 mM 4-methylumbelliferyl-phosphate in a solution of 100 mM AMP, 1.0mM MgCl₂, 0.1% NaN₃, and 4.0 mM tetramisole at pH 10.3) was added to thesolid phase. The resulting rate of fluorescence was measured. Theresults of the assay are summarized in Tables 26 and 27. A comparison ofcapture techniques was performed by repeating the above described assayusing Rubella virus and virus-coated microparticles as reagents to beimmobilized on a glass fiber matrix treated with a polymeric quaternaryammonium compound, and on an untreated glass fiber matrix.

                  TABLE 26    ______________________________________    Rubella Assay    Untreated Glass Fiber Matrix                Rate of fluorescence (counts/sec/sec)    Anti-Rubella IgG         Virus-coated    (IU/ml)       Rubella Virus                             Microparticles    ______________________________________    0             176        182    15            261        395    50            279        911    100           334        1396    200           391        2141    500           649        2670    ______________________________________

                  TABLE 27    ______________________________________    Rubella Assay    Treated Glass Fiber Matrix                Rate of fluorescence (counts/sec/sec)    Anti-Rubella IgG         Virus-coated    (IU/ml)       Rubella Virus                             Microparticles    ______________________________________    0             131        184    15            263        396    50            672        867    100           1161       1328    200           1851       2212    500           3518       2821    ______________________________________

The amount of Rubella virus used as the capture reagent in each test wasthe same as that used for coating the microparticles.

Example 26 Rubella antibody Detection Using an Ion-Capture SandwichAssay with Directly Detectable Labels

The solid phase devices and Rubella virus were prepared substantially inaccordance with the processes described in Example 25, above. Theindicator reagent was a specific binding member for the gamma chain ofhuman IgG labeled with either a gold or selenium compound.

Test solutions were prepared by combining the diluted Rubella virus (100μl) with three drops of either a positive or negative standard reagent.The positive reagent was human serum positive for anti-Rubella IgG, andthe negative reagent was human serum negative for anti-Rubella IgG. Thetest solutions were mixed and incubated at room temperature for oneminute. The test solutions were then applied to the solid phase. Eachsolid phase was rinsed with buffer (100 μl), and indicator reagent (50μl) was added. Each solid phase was then rinsed two time with buffer(150 μl).

A dark purple color was detected on the solid phase material for thepositive test, while no color was detected for the negative test, whenthe gold-labeled indicator reagent was used. A dark pink color wasdetected on the solid phase material for the positive test, while alight pink color was detected for the negative test, when theselenium-labeled indicator reagent was used.

It will be appreciated by one skilled-in-the-art that the concepts ofthe present invention are equally applicable to any separationtechniques or homogeneous binding assays (wherein the signal generatingability of the label is not altered during the binding reaction) byusing oppositely charged solid phase materials and capture reagents. Theembodiments described in detail herein are intended as examples ratherthan as limitations of the polyelectrolyte reactions and assays. Thus,the description of the invention is not intended to limit the inventionto the particular embodiments described, but it is intended to encompassall equivalents and subject matter within the spirit and scope of theinvention as described above and as set forth in the following claims.

What is claimed is:
 1. A method for determining the presence or amountof Rubella antibody in a test sample, comprising the steps of:a)providing a test sample suspected of containing Rubella antibody; b)forming a reaction mixture by contacting the test sample with a solublecapture reagent which comprises unbound Rubella virus; c) incubating thereaction mixture under conditions sufficient for the Rubella antibodyand capture reagent to react and form soluble capture reagent/Rubellaantibody complexes; d) after incubating, determining the presence oramount of Rubella antibody by(i) contacting the reaction mixture with asolid phase containing a reaction site comprising a polymeric cationsubstance imparting a net positive charge to the solid phase so thatcapture reagent/Rubella antibody complexes and unreacted capture reagentare separated from the reaction mixture and coupled to the solid phaseby the ionic attraction of the capture reagent and solid phase, (ii)contacting said solid phase with an indicator reagent which comprises aspecific binding member for Rubella antibody and a detectable label,(iii) incubating the solid phase and the indicator reagent underconditions sufficient for the capture reagent/Rubella antibody complexesand indicator reagent to react and form capture reagent/Rubellaantibody/indicator reagent complexes, and (iv) detecting labelassociated with the solid phase or with the unreacted indicator reagent.2. The method according to claim 1, wherein said polymeric cationsubstance is a polymeric quaternary ammonium compound.
 3. The methodaccording to claim 2, wherein said polymeric cation substance is ahomopolymer of dimethyidiallylammonium chloride.
 4. The method accordingto claim 1, wherein said specific binding member is an anti-human IgGantibody.
 5. The method according to claim 1, wherein said specificbinding member is an anti-human IgM antibody.
 6. A method fordetermining the presence or amount of Rubella antibody in a test sample,comprising the steps of:a) providing a test sample suspected ofcontaining Rubella antibody; b) forming a reaction mixture by contactingthe test sample with a soluble capture reagent which comprises unboundRubella virus; c) incubating the reaction mixture under conditionssufficient for the Rubella antibody and capture reagent to react andform soluble capture reagent/Rubella antibody complexes; d) afterincubating, determining the presence or amount of Rubella antibody by(i)contacting the reaction mixture with a solid phase containing a reactionsite comprising a polymeric cation substance imparting a net positivecharge to the solid phase so that capture reagent/Rubella antibodycomplexes and unreacted capture reagent are separated from the reactionmixture and coupled to the solid phase by the ionic attraction of thecapture reagent and solid phase, (ii) contacting said solid phase withan ancillary binding member comprising a specific binding member forRubella antibody, and an indicator reagent comprising a specific bindingmember for said ancillary binding member and a detectable label, (iii)incubating the solid phase with said ancillary binding member and saidindicator reagent, thereby immobilizing said indicator reagent on saidsolid phase in proportion to the amount of Rubella antibody present inthe test sample, and (iv) detecting label associated with the solidphase or with the unreacted indicator reagent.
 7. The method accordingto claim 6, wherein said polymeric cation substance is a polymericquaternary ammonium compound.
 8. The method according to claim 7,wherein said polymeric cation substance is a homopolymer ofdimethyldiallylammonium chloride.
 9. The method according to claim 6,wherein said ancillary binding member is an anti-human IgG antibody. 10.The method according to claim 6, wherein said ancillary binding memberis an anti-human IgM antibody.
 11. The method according to claim 6,wherein said ancillary binding member is a biotinylated anti-human IgGantibody and said indicator reagent is a labeled binding member specificfor biotin.
 12. The method according to claim 6, wherein said ancillarybinding member is a biotinylated anti-human IgM antibody and saidindicator reagent is a labeled binding member specific for biotin.